Sealing or filling tissue defects using polyfunctional crosslinking agents and protein polymers

ABSTRACT

Proteinaceous polymers having repetitive units from naturally occurring structural proteins are employed as backbones for functionalities for crosslinking to provide strongly adherent tissue adhesive compositions for bonding together separated tissue, and for sealing or filling tissue defects by injecting the compositions into the defects. Particularly, block copolymers having repeating units of elastin and fibroin are employed having lysine substitutions in spaced apart units, where the amino group can be crosslinked using difunctional crosslinking agents such as glutaraldehyde, activated diolefins, diisocyanates, acid anhydrides or diamines. The protein polymer contains at least 40 weight percent of repetitive units of 3 to 30 amino acids, preferably 3 to 15 amino acids, of at least one naturally occurring structural protein and at least two amino acids containing a functional group capable of reacting with the crosslinking agent. The protein polymer generally has a molecular weight of at least about 30 kD and not more than 250 kD. A preferred protein polymer contains at least 70 weight percent of repetitive units of Gly-Ala-Gly-Ala-Gly-Ser and Gly-Val-Gly-Val-Pro, where in at least two units an amino acid is substituted with one of lysine or arginine, and the protein polymer has a lysine and arginine equivalent weight in the range of 1 to 20 kD and contains at least two amino acids having a functional group capable of reacting with at least one of aldehyde, isocyanate, thioisocyanate and activated carboxy. The protein polymer is produced by recombinant DNA technology, and a kit may be formed containing the crosslinking agent and protein polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/642,246,filed May 2, 1996, now U.S. Pat. No. 6,033,654, which is acontinuation-in-part of application Ser. No. 08/435,641, filed May 5,1995, now U.S. Pat. No. 5,817,303, whose disclosure is incorporatedherein by reference.

INTRODUCTION

1. Technical Field

The field of this invention is physiologically acceptable compositionsfor use as tissue adhesives and sealants.

2. Background

In many situations, there is a need to bond separated tissues. Suturesand staples are effective and well established wound closure devices.However, there are surgical procedures where classical repair proceduresare unsatisfactory, limited to highly trained specialists (e.g.microsurgery), or not applicable due to tissue or organ fragility,inaccessibility (e.g. endoscopy procedures), or fluid loss, includingcapillary “weeping”. Tissue adhesives and sealants have been developedto meet these needs. They may be used to seal or reinforce wounds thathave been sutured or stapled, as well as finding independent use. Theleading commercial products are fibrin glues and cyanoacrylates.However, both products have significant limitations which have preventedtheir widespread use.

Cyanoacrylates are mainly used for cutaneous wound closure in facial andreconstructive surgery. The appeal of cyanoacrylates is their speed ofbonding, which is almost immediate, and its great bond strength.However, its speed of bonding can be a disadvantage, since glued tissuemust be cut again in order to reshape it to the desired conformation.Additionally, it can only be used on dry substrates since its mode ofaction is through a mechanical interlock, limiting its use as a sealant,and it is relatively inflexible compared to surrounding tissue.Cyanoacrylates are also known to be toxic to some tissues and althoughit is not considered to be biodegradable, potential degradation productsare suspected to be carcinogenic.

Fibrin glues comprising blood-derived fibrinogen, factor-XIII andthrombin function primarily as a sealant and hemostat and have been usedin many different surgical procedures within the body. They have beenshown to be non-toxic, biocompatible and biodegradable. They are able tocontrol excessive bleeding and decrease fibrosis. However, tissuesbonded with fibrin cannot be subjected to even moderate tensile stresswithout rupturing the bond. It takes about three to ten minutes for aninitial bond to develop, but requires about 30 minutes to several hoursfor full strength to develop. Depending upon the application, theproduct may also resorb too quickly. Use of recombinantly producedfibrinogen, factor XIII, thrombin and related components (e.g. fibrin,activated factor XIII) has not been demonstrated to improve the settingtime or strength of fibrin glues. Fibrin glues derived fromheterologous, human and animal, serum may provoke undesirable immuneresponses, and expose the patient to the potential risk of viralinfection. Autologous fibrin glues may be impractical to obtain and useand may compromise patient safety.

There is, therefore, substantial interest in developing products whichhave the biocompatibility of fibrin glues, but which set more quicklyand have enhanced strength. These products should be readily available,desirably from other than natural sources, be easily administered andcapable of resorption over time.

Relevant Literature

Tissue adhesives are described in: Tissue Adhesives in Surgery,Matsumoto, T., Medical Examination Publishing Co., Inc. 1972 and Sierra,D. H., J. Biomat. App. 7:309-352, 1993. Methods of preparation ofprotein polymers having blocks of repetitive units are described in U.S.Pat. No. 5,243,038 and EPA 89.913054.3.

SUMMARY OF THE INVENTION

Polymeric compositions and methods for their use are provided, where thepolymeric compositions are capable of in situ chemical crosslinking toprovide novel crosslinked polymeric products, which have good mechanicaland biological properties, as exemplified by strong adherent bonds totissue. The compositions can be used in a variety of applicationsrelated to their physical, chemical and biological properties, to bondto separated tissue to provide at least one of the characteristics of astable, flexible, resorbable bond.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The subject compositions comprise high molecular weight recombinantpolymers having one or a combination of repeating units related tonaturally occurring structural proteins. Of particular interest are therepeating units of fibroin, elastin, collagen, and keratin, particularlycollagen and combinations of fibroin and elastin. The polymers havefunctional groups which can be chemically crosslinked underphysiological conditions with physiologically acceptable crosslinkers,so as to form a composition which has strong adherent properties to avariety of substrates, has strong mechanical properties in maintainingthe joint between the substrates, and can be formulated to have goodresorption properties.

Of particular interest, the subject compositions provide stronglyadherent bonds to tissue to maintain separated tissue in a contiguousspacial relationship. The subject compositions may also be employed assealants, where the compositions may serve to fill a defect void intissue, to augment tissue mass or bond synthetic materials to tissues.The subject compositions may also serve as depots in vivo by being mixedwith a drug composition, either when used as an adhesive for bondingtissue together or for other bonding or solely as a slow release sourceof the drug.

The functionalities for crosslinking may be all the same or combinationsof functionalities and may include the functionalities of naturallyoccurring amino acids, such as amino, e.g. lysine, carboxyl, e.g.aspartate and glutamate, guanidine, e.g. arginine, hydroxyl, e.g. serineand threonine, and thiol, e.g. cysteine. Preferably, the functionalityis amino (including guanidine).

The polymers will have molecular weights of at least about 15 kD,generally at least about 30 kD, preferably at least about 50 kD andusually not more than 250 kD, more usually not more than about 150 kD.The polymers will have at least two functionalities, more usually atleast about four functionalities, generally having an equivalent weightper functionality in the range of about 1 kD to 40 kD, more usually inthe range of about 3 kD to 20 kD, preferably in the range of about 3kDto 10 kD, there being at least 3, usually at least 6, functionalitiesavailable for crosslinking. If desired, one may use mixtures ofpolymers, where the polymers have combinations of functionalities orhave different functionalities present e.g. carboxyl and amino, thioland aldehyde, hydroxyl and amino, etc. Thus, depending upon thefunctionalities and the crosslinking agent, one can form amides, imines,ureas, esters, ethers, urethanes, thioethers, disulfides, and the like.

The individual units in the polymer may be selected from fibroin, GAGAGS(SEQ ID NO:01); elastin, GVGVP (SEQ ID NO:02); collagen GXX, where theX's may be the same or different, and at least 10 number % and not morethan 60 number % of the X's are proline, and keratin, AKLK/ELAE (SEQ IDNO:3). The desired functionality may be substituted for one of the aminoacids of an individual unit or be present as an individual amino acid orpart of an intervening group of not more than about 30 amino acids,usually not more than about 16 amino acids. In the former case, within ablock of repeats, one or more of the repeats is modified to introduce acrosslinking functionality which would otherwise not normally bepresent. Thus a valine may be replaced with a lysine, a glycine with anarginine, an alanine with a serine, and the like. In the latter case,there would be an intervening functionality between a block of repeatunits, where the number of intervening functionalities would be based onthe ranges indicated previously.

Of particular interest are copolymers, either block or random,preferably block, where in the case of elastin and fibroin, the ratio ofelastin units to fibroin units is in the range of 16-1:1, preferably8-1:1, where blocks may have different ratios. Normally, in blockcopolymers, each block will have at least two units and not more thanabout 32 units, usually not more than about 24 units. By substituting anamino acid in the unit with an amino acid having the appropriatefunctionality, one can provide for the appropriate number offunctionalities present in the polymer or employ intervening groupsbetween blocks.

The individual amino acid repeat units will have from about 3 to 30amino acids, usually 3 to 25 amino acids, more usually 3 to 15 aminoacids, particularly 3 to 12 amino acids, more particularly about 3 to 9amino acids. At least 40 weight %, usually at least 50 weight %, moreusually at least 70 weight %, of the protein polymer will be composed ofsegments of repetitive units containing at least 2 identical contiguousrepetitive units. Generally repeat blocks will comprise at least 2, 4, 7or 8 units, and combinations thereof, where copolymers are employed,where the unit which is modified with the crosslinking functionality iscounted as a unit.

While for the most part, the polymers of the subject invention will havethe active functionality of a naturally occurring amino acid in thechain of the polymer, if desired, pendent groups may be employed toprovide the desired functionalities. For example, carboxyl groups may bereacted with polyamines so as to exchange a carboxyl functionality for asingle amino or plurality of amino groups. An amino group may besubstituted with a polycarboxylic acid, so that the amino group will bereplaced with a plurality of carboxylic groups. A thiol may be replacedwith an aldehyde, by reaction with an aldehydic olefin, e.g. acrolein,so as to provide for an aldehyde functionality. Other functionalitieswhich may be introduced, if desired, include phosphate esters, activatedolefins, e.g. maleimido, thioisocyanato, and the like. Thefunctionalities may be greatly varied from those which naturally occurto provide opportunities for crosslinking. In some instances, this maybe desirable to increase the number of functionalities per unitmolecular weight, while not increasing the number of functionalitiesalong the chain, for replacing one functionality with another, e.g.thiol with aldehyde, allowing for greater variation in the choice ofcrosslinking agent.

The crosslinking agent will normally be difunctional, where thefunctionalities may be the same or different, although higherfunctionality may be present, usually not exceeding fourfunctionalities. Depending upon the particular functionalities availableon the polymers, various crosslinking agents may be employed. Thecrosslinking agents will usually be at least about three carbon atomsand not more than about 50 carbon atoms, generally ranging from about 3to 30 carbon atoms, more usually from about 3 to 16 carbon atoms. Thechain joining the two functionalities will be at least one atom and notmore than about 100 atoms, usually not more than about 60 atoms,preferably not more than about 40 atoms, particularly not more thanabout 20 atoms, where the atoms may be carbon, oxygen, nitrogen; sulfur,phosphorous, or the like. The linking group may be aliphaticallysaturated or unsaturated, preferably aliphatic, and may include suchfunctionalities as oxy, ester, amide, thioether, amino, and phosphorousester. The crosslinking group may be hydrophobic or hydrophilic.

Various reactive functionalities may be employed, such as aldehyde,isocyanate, mixed carboxylic acid anhydride, e.g. ethoxycarbonylanhydride, activated olefin, activated halo, amino, and the like. Byappropriate choice of the functionalities on the protein polymer, andthe crosslinking agent, rate of reaction and degree of crosslinking canbe controlled.

Various crosslinking agents may be employed, particularly those whichhave been used previously and have been found to be physiologicallyacceptable. Crosslinking agents which may be used include dialdehydes,such as glutaraldehyde, activated diolefins, diisocyanates such as,tetramethylene diisocyanate, hexamethylene diisocyanate, octamethylenediisocyanate, acid anhydrides, such as succinic acid dianhydride,ethylene diamine tetraacetic acid dianhydride, diamines, such ashexamethylene diamine, cyclo(L-lysyl-L-lysine), etc. The crosslinkingagent may also contain unsymmetrical functionalities, for example,activated olefin aldehydes, e.g. acrolein and quinoid aldehydes,activated halocarboxylic acid anhydride, and the like. The crosslinkingagents will usually be commercially available or may be readilysynthesized in accordance with conventional ways, either prior toapplication of the adhesive or by synthesis in situ.

In some instances it may be desirable to react a physiologicallyacceptable second compound, which serves as a modifying unit, with apolyfunctional, usually bifunctional, compound to change the nature ofthe crosslinking. The addition of the second compound may be to enhancethe rate of crosslinking, change the solubility properties of thecrosslinker, enhance or reduce the strength of the crosslinked polymer,enhance or reduce the resorption rate, or provide for other physical,chemical or biological properties of interest. The polyfunctional secondcompound may be reacted with the crosslinking compound prior to reactionwith the protein or concurrently with the reaction with the protein.Where the reaction is prior, the resulting crosslinking product will bephysiologically acceptable and when concurrent, the polyfunctionalsecond compound, the crosslinking compound and the resultingcrosslinking product will be physiologically acceptable, when used invivo. The ratio of the polyfunctional second compound to thecrosslinking compound will generally be in the range of about 0.1-2:1,more usually in the range of about 0.1-1:1, depending on the reactivityof the polyfunctional second compound when the polyfunctional secondcompound and crosslinking agent are brought together, the number ofcrosslinks desired in the final protein composition, the size of thebridge between protein molecules, and the like.

The nature of the polyfunctional second compound may vary widely. Thefunctional groups present may be the same or different from thefunctional groups present on the polymer, but will be reactive with thefunctionalities of the crosslinking compound. For example, thepolyfunctional second compound may have amino and/or hydroxyl groups,where the protein has amino or hydroxyl functionalities. By employing adiisocyanate with a diol, diurethanes will be produced. Thus, the chaincrosslinking the proteins will comprise 2 or more urethanes.

In many instances, the polyfunctional second compound will include aninternal functionality that does not participate in the reaction, butprovides various other characteristics to the crosslinking agent or thecrosslinked protein product. Characteristics of interest includehydrophilicity, hydrolytic instability, sensitivity to enzymaticdegradation, biocompatibility, shear strength, and the like. For themost part internal functionalities will comprise oxygen, sulfur andnitrogen atoms, such as ethers, carboxylic acid esters, includingurethanes, amino groups, amides, ketones, dithiols and the like. Toenhance the rate of resorption, ester groups are of interest, while toenhance hydrophilicity, the same groups maybe employed as well asethers, such as polyoxyalkylene groups.

The polyfunctional second compound will generally have at least 2 carbonatoms and not more than 50 carbon atoms, usually not more than about 30carbon atoms, desirably having not more than about 16 carbon atoms perheteroatom. Naturally occurring or synthetic bifunctional compounds maybe employed. Illustrative compounds include lysine, arginine,di-(2′-aminoethyl) malonate, citrate, lysyl lysine, 2′-aminoethylglycinate, O, N-diglycinyl ethanolamine, diethylene glycol diglycinate,cystine, and the like. To provide terminal amino groups, various lowmolecular weight amino acids may be used, particularly glycine andalanine bonded to an intervening difunctional compound, such as ethyleneglycol, diethylene glycol, and tetraethylene glycol, propanediol,1,4-butyn-2-diol, ascorbic acid, etc.

The subject compositions may be prepared prior to the use of theadhesive by combining the protein polymer and the crosslinking agent,where one or both may have extenders. The two compositions may bereadily mixed in accordance with conventional ways, for example, usingsyringes which can inject the ingredients into a central reactor and themixture mixed by drawing the mixture back into the syringes and movingthe mixture back and forth. Alternatively, the two compositions may bedispensed simultaneously at the site of application. In some instancesit may be desirable to allow the crosslinking agent to partially reactwith the protein prior to adding the polyfunctional second compound.Alternatively, one may mix the polyfunctional second compound with theprotein prior to mixing with the crosslinking agent.

Usually, the polymer will be available as a dispersion or solution,particularly aqueous, generally the concentration of the protein polymerbeing in the range of about 50 mg to 1 g/ml, more usually from about 100to 800 mg/ml. The solution may be buffered at a pH which enhances orretards the rate of crosslinking. Usually the pH will be in the range ofabout 2 to 12, more usually 8 to 11. Various buffers may be used, suchas phosphate, borate, carbonate, etc. The cation can have an effect onthe nature of the product, and to that extent, the alkali metalspotassium and sodium, are preferred. The protein composition willgenerally be about 5 to 40, more usually from about 5 to 20, preferablyfrom about 10 to 20 weight %, to provide for a composition which may bereadily handled, will set up within the desired time limit, and thelike. The buffer concentration will generally be in the range of about50 to 500 mM. Other agents may be present in the protein solution, suchas stabilizers, surfactants, and the like. If the polyfunctional secondcompound is present its concentration will be determined in accordancewith its ratio to the crosslinking agent and the polymer.

The ratio of crosslinking agent to polymer will vary widely, dependingupon the crosslinking agent, the number of functionalities present onthe polymer, the desired rate of curing, and the like. Generally, theweight ratio of polymer to crosslinking agent will be at least about 1:1and not greater than about 100:1, usually not greater than about 50:1,generally being in the range of about 2 to 50:1, but in some instancesmay not be more than 30:1. The equivalent ratio of protein tocrosslinking agent will generally be in the range of about 0.1-1:3, moreusually in the range of about 0.5-2:2. Considerations in selecting theprotein-crosslinking agent equivalent ratio will be the rate of setup,reactivity of the crosslinking agent, relative solubility of thecrosslinking agent in the mixture, physiological properties of thecrosslinking agent, desired degree of stability of the crosslinkedproduct, and the like.

If desired, various extenders or extending agents may be used,particularly naturally occurring proteins. Such extenders will usuallynot exceed 50 weight percent of the composition, generally not exceedingabout 20 weight percent, more usually not exceeding about 10 weightpercent. Extenders which may be employed include, but are not limitedto: synthetic polymers, both addition and condensation polymers, bothprotein and non-protein, such as polylactides, polyglycolides,polyanhydrides, polyorthoesters, polyvinyl compounds, polyolefins,polyacrylates, polyethylene glycol, polyesters, polyvinyl alcohol,polyethers, copolymers and derivatives thereof; and naturally occurringpolymers, such as proteins and non-proteins, including collagen,fibrinogen, fibronectin, laminin, keratin, chitosan, heparin, dextran,alginates, cellulose, glycosoaminoglycans, hyaluronic acid,polysaccharides, derivatives thereof, and the like. The extenders maymodulate the setting time and provide for desirable physical orphysiological properties of the adhesive.

Based on the lap shear tensile strength test described in theexperimental section, within 30 minutes, usually within 15 minutes, moreusually within 5 minutes, the lap shear tensile strength will be atleast 100, preferably at least about 250, more preferably at least about300, usually not exceeding about 4000, more usually not exceeding about3000 g/cm².

The subject compositions may be applied to the tissue in any convenientway, for example by using a syringe, catheter, cannula, manuallyapplying the composition, spraying or the like. The subject compositionsmay be applied to the tissue prior to or during the time the tissuesegments are held in contiguous relationship. The subject compositionswill rapidly develop substantial shear strength, so as to maintain thetissue in proximity. In some situations there will be an interest inhaving the composition decompose after some reasonable period of time,usually at least one week and generally not more than about four weeks.

Tissues of interest include vascular vessels such as an artery, vein orcapillary, muscel, nerve, organs, e.g. liver, spleen, etc., lung, dura,colon, and the like.

In addition to their use as adhesives, the subject compositions may beused to seal or fill defects, e.g. voids or holes, in tissue, andtherefore find use as sealants. Thus, the compositions may serve to stopor staunch the flow of fluid, e.g. blood, through ruptured vessels, e.g.arteries, veins, capillaries and the like. In using the subjectcompositions as sealants, the composition will be applied, as describedabove, at the site of the defect, whereby it will set and seal thedefect. The compositions may be injected into normal or abnormal tissuesto augment the tissue mass, e.g. dermis.

The subject compositions may also find use in the formation of articlesof manufacture, by themselves or in combination with other materials. Inone application, articles may be produced for use internally to amammalian host, where there is an interest in biocompatibility,reabsorption rate, ability to vascularize, tissue adhesive and/orbonding capability, and the like. Various articles can be prepared, suchas gels, films, threads, coatings, formed objects such as pins andscrews, or injectable compositions which are flowable, where theinjectable composition may set up and bond or seal tissues, form a depotfor a drug, augment tissue or be a filler, coating or the like. Theformed objects may be prepared in accordance with conventional ways,such as molding, extrusion, precipitation from a solvent, solventevaporation, and the like. The flowable depot can be obtained by using amolecular dispersion, fine particles in a medium saturated with apolymer, using a melt, where the melting temperature may be achieved byadding physiologically acceptable additives, and the like.

The articles may find use in a variety of situations associated with theimplantation of the article into a mammalian host or the application ofthe article to the surface of a mammalian host, e.g. wound healing, burndressing, etc. Those situations, where the performance of the articlesmay be retained for a predetermined time and replaced by naturalmaterials through natural processes, desirably employ materials whichwill be resorbed after having fulfilled their function in maintainingtheir role until the natural process has reestablished a naturalstructure. Thus, the compositions may find use in holding tissuetogether, covering tissue, encapsulating cells for organs, providing acoating that cells can invade and replace the composition with naturalcomposition, e.g., bone, soft tissues and the like.

To enhance the rate of curing of the polymeric composition, thecomposition may be partially prepolymerized. When prepolymerized, thepolymer will usually have at least about 3% of the total number ofcrosslinks and not more than about 75% of the total number ofcrosslinks, as compared to completion of the crosslinking action. Thenumber of crosslinks should allow the resulting product to be workableand provide sufficient time prior to set up for it to be manipulated andused. Alternatively, one may react the functional groups with an excessof the crosslinking reagent, so that the effect is to substitute thefunctionality of the protein with the functionality of the crosslinkingagent. The protein with the substituted functionality may then be usedto crosslink protein with the original functionality or with apolyfunctional second compound.

The subject compositions may also be used as depots to provide for arelatively uniform release of a physiologically active product, e.g., adrug. The drug may be mixed with a subject composition at an appropriateconcentration prior to crosslinking. As the crosslinked polymer isdegraded, the drug will be released due to diffusion as well as erosionof the external surface of the depot. By controlling the form or shapeof the depot, the degree of crosslinking, the concentration of the drugand the like, a physiologically therapeutic level of the drug may bemaintained over extended periods of time. The period required forabsorption can be as short as 0.5 day and may exceed 4, 6 or 8 weeks ormore, depending upon the particular composition and the application.

The protein polymer compositions may be prepared in accordance withconventional ways. See, for example, U.S. Pat. No. 5,243,038, whichdisclosure is incorporated herein by reference. Briefly, sequences maybe synthesized comprising a plurality of repeating units, wherecomplementary sequences result in dsDNA having overhangs. A series ofdsDNA molecules may be prepared and stepwise introduced into a cloningvector as the gene for the protein is constructed. A monomer can beobtained in this way, which may be sequenced to ensure that there havebeen no changes in the sequence, followed by multimerization of themonomer, cloning and expression. For further details, see the aboveindicated patent.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1

Methods

The construction of synthetic DNA and its use in large polypeptidesynthesis is described in U.S. Pat. No. 5,243,038; PCT/US89/05016 andPCT/US92/09485, the disclosures of which are herein incorporated byreference. Modifications to these methods and additional methods usedare described below.

1. Use of Filters and Columns for DNA Purification

A. Ultrafree®-Probind filter unit (“Probind”, Millipore): the DNAcontaining solution was applied to the filter unit and spun at 12,000RPM for 30 seconds in a Sorvall Microspin 24S.

B. Microcon-30 filter (Amicon): the DNA containing solution was washedby applying to the filter and exchanging twice with H₂O by spinning at12,000 RPM for 6 minutes in a microfuge.

C. Bio-Spin 6 column (“Bio-Spin”, BioRad): Salts and glycerol wereremoved from the DNA solution by applying to the column, previouslyequilibrated in TEAB (triethyl ammonium bicarbonate pH 7.0), andspinning in a Sorvall RC5B centrifuge using an HB4 rotor at 2,500 RPMfor 4 minutes.

2. Phosphatase Treatment of DNA

Phosphatase treatment of DNA was also performed by resuspending ethanolprecipitated DNA from the restriction enzyme digest in 20 mM Tris-HCl pH8.0, 10 mM MgCl₂ to a final DNA concentration of 20 μg/ml. ShrimpAlkaline Phosphatase (SAP) was added at 2 U/μg of DNA and the mixturewas incubated at 37° C. for one hour, heat inactivated for 20 minutes at65° C. and then passed through a Probind filter and subsequently aBio-Spin column.

3. Preparative Agarose Gel Electrophoresis

For agarose ligation, the buffer used was 1×TAE (50 mM Tris-acetate, pH7.8).

4. Agarose DNA Ligation

The agarose was melted at 65° C., the temperature was then lowered to37° C. and ligation buffer (5×=100 mM Tris-HCl, pH 7.5, 50 mM MgCl₂, 50mM DTT, 1 mM ATP) was added; the tube was then placed at roomtemperature and ligase was added (1000 units T4 DNA ligase (NEB)). Thereaction volume was usually 50 μl. The reaction was incubated at 15° C.for 16-18 hours.

5. Agarose DNA Purification Using an Ultrafree®-MC Filter Unit

This procedure can be used for agarose slices up to 400 μl in size.After agarose gel electrophoresis, the DNA is visualized by ethidiumbromide staining and the agarose block containing the DNA band ofinterest is excised. The agarose is then frozen at −20° C. for 1 hour,then quickly thawed at 37° C. for 5 minutes. The agarose is thenthoroughly macerated. The pieces are then transferred into the samplecup of the filter unit and spun at 5,000×g in a standard microfuge for20 minutes. The agarose is then resuspended in 200 μl of Tris-EDTA, orother buffer, and incubated at room temperature for 30 minutes to allowfor elution of additional DNA from the gel. The mixture is thencentrifuged for an additional 20 minutes at 10,000 RPM. The DNA is, atthis point, in the filtrate tube separated from the agarose fragmentsand ready for subsequent DNA manipulations.

6. Preparation of Antibody to Artificially Synthesized Peptides

The same procedures were used as described in U.S. Pat. No. 5,243,038,PCT/US89/05016 and PCT/US92/09485.

7. Immunoblotting of Proteins in Gels

An alternative to the ¹²⁵I-Protein A detection method was used. Thismethod relied on a chemiluminescent signal activated by horseradishperoxidase (HRP). The chemiluminescent reagents are readily availablefrom several suppliers such as Amersham and DuPont NEN. The western blotwas prepared and blocked with BLOTTO. A number of methods were used tointroduce the HRP reporter enzyme including, for example, ahapten/anti-hapten-HRP, a biotinylated antibody/streptavidin-HRP, asecondary reporter such as a goat or mouse anti-rabbitIgG-biotinylated/streptavidin-HRP, or a goat or mouse-anti rabbitIgG-HRP. These reagents were bought from different sources such asBioRad or Amersham and occasionally biotinylated antibodies wereprepared in our laboratory using Biotin NHS from Vector Laboratories,Burlingame, Calif. (Cat. #SP-1200) following the procedure accompanyingthe product. The following is an example of a procedure used to detectthe expression of protein polymers.

The blot was placed in 15 ml of BLOTTO solution containing biotinylatedgoat anti-rabbit IgG (BioRad) diluted in BLOTTO (1:7500) and gentlyagitated for 2 hours at room temperature. The filter was then washed for30 minutes with 3 changes of TSA (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 0.2%sodium azide) and then for 5 minutes each in TBS with 0.1% TWEEN®20. Theblot was then incubated for 20 minutes at room temperature with gentlerotation, in 20 ml of TBS (100 mM Tris Base, 150 mM NaCl, pH 7.5)HRP-Streptavidin (Amersham) diluted 1:1000 in TBS with 0.1% Tween 20.The blot was then washed three times for 5 minutes each in TBS with 0.3%Tween 20 and then three times for 5 minutes each in TBS with 0. 1% Tween20. The blot was then incubated for 1 minute with gentle agitation in 12ml of development solutions #1 an #2 (Amersham) equally mixed. The blotwas removed from the development solution and autoradiographed.

8. Protein Expression Analysis

An overnight culture which had been grown at 30° C. was used toinoculate 50 ml of LB media contained in a 250 ml flask. Kanamycin wasadded at a final concentration of 50 μg per ml and the culture wasincubated with agitation (200 rpm) at 30° C. When the culture reached anOD₆₀₀ of 0.8, 40 ml were transferred to a new flask prewarmed at 42° C.and incubated at the same temperature for approximately 2 hours. Thecultures (30° and 42°) were chilled on ice and OD₆₀₀ was taken. Cellswere collected by centrifugation and then divided in 1.0 OD₆₀₀ aliquotsand used to perform western analysis using the appropriate antibodies.

9. Amino Acid Analysis

Amino acid derivatives were analyzed by reverse phase HPLC using aWaters 600E system.

10. Peptide Synthesis

Synthetic peptides were also prepared on a Rainin/Protein TechnologiesPS3 FMOC peptide synthesizer. Both the synthesis and cleavage wereaccomplished using the methods supplied by the manufacturer in theinstrument manual.

11. In vitro DNA Synthesis

The β-cyanoethyl phosphoramidites, controlled-pore glass columns and allsynthesis reagents were obtained from Applied Biosystems, Foster City,Calif. Synthetic oligonucleotides were prepared by the phosphitetriester method with an Applied Biosystems Model 381A DNA synthesizerusing a 10-fold excess of protected phosphoramidites and 0.2 μmole ofnucleotide bound to the synthesis support column. The chemistries usedfor synthesis are the standard protocols recommended for use with thesynthesizer and have been described (Matteucci et al., J. Amer. Chem.Soc., 103:3185-3319 (1981)). Deprotection and cleavage of the oligomersfrom the solid support were performed according to standard proceduresas provided by Applied Biosystems. The repetitive yield of the synthesisas measured by the optical density of the removed protecting group asrecommended by Applied Biosystems was greater than 97.5%.

The crude oligonucleotide mixture was purified by preparative gelelectrophoresis as described by the Applied Biosystems protocols inEvaluating and Isolating Synthetic Oligonucleotides, 1992 (Formerly:User Bulletin 13, 1987). The acrylamide gel concentration varied from 10to 20% depending upon the length of the oligomer. If necessary, thepurified oligomer was identified by UV shadowing, excised from the geland extracted by the crush and soak procedure (Smith, Methods inEnzymology, 65:371-379 (1980)).

For DNA synthesis of oligonucleotides longer then 100 bases, thesynthesis cycle was changed from the protocol recommended by AppliedBiosystems for the 381A DNA synthesizer. All the reagents used werefresh. All the reagents were supplied by Applied Biosystems except forthe acetonitrile (Burdick and Jackson Cat #017-4 with water content lessthen 0.001%) and the 2000 Å pore size column (Glen Research). Due to thelength of the oligo, interrupt pauses had to be inserted during thesynthesis to allow changing the reagent bottles that emptied duringsynthesis. This interrupt pause was done at the cycle entry step and thepause was kept as short as possible. The washes after detritylation byTCA, through the beginning of each synthesis cycle, were increased fromabout 2× to 3× over the recommended time. The time allocated for thecapping was also increased to limit truncated failure sequences. Afterthe synthesis the deprotection was done at 55° C. for 6 hours. Afterdesalting the synthesized DNA was amplified using PCR.

12. Sequencing of DNA

Storage and analysis of data utilized software from DNA Strider, DNAInspection IIe or DNAid for Apple Macintosh personal computer.

13. Dideoxy DNA Sequencing of Double Stranded Plasmid DNA

As described in U.S. Pat. No. 5,243,038, plasmid DNA was prepared on asmall scale. Primers were synthesized using a DNA synthesizer and wereannealed to the plasmid DNA following the procedure described for M13sequencing. The sequencing reactions were done using Sequenase (UnitedStates Biochemicals) and the conditions were as recommended by thesupplier. All sequences were run on polyacrylamide gels.

14. PCR Amplification

The PCR reaction was performed in a 100 μl volume in a Perkin Elmerthin-walled Gene Amp™ reaction tube. Approximately 1 μM of each primerDNA was added to 1×PCR buffer (supplied by Perkin Elmer as 10×solution),200 μM of each dNT, 5U AmpliTaq, and several concentrations of thetarget DNA. Amplification was performed in a Perkin Elmer DNA Thermalcycler model 480 for 30 cycles with the following step cycles of 12minutes each: 95° C., 62° C., and 72° C. Aliquots from the differentreactions were analyzed by agarose gel electrophoresis using 1.5% lowmelting point agarose in 0.5×TA buffer. The reaction mixtures that gavethe desired band were pooled and spun through a Probind filter to removethe AmpliTaq enzyme, then a Microcon-30 filter and a Bio-Spin column.The DNA was then concentrated in vacuo.

15. Diamine Synthesis

2-Aminoethyl Glycinate

Concentrated sulfuric acid (9.90 g, 0.101 mole) was diluted into 10 mLof water. Glycine (7.50 g, 0.100 mole), 2-aminoethanol (6. 10 g, 0.100mole) and the diluted sulfuric acid were placed in a 250 mL, 3-neck,round bottom flask fitted with a stopper, a mechanical stirrer, aheating mantle, and a Dean-Stark water trap. The contents of theapparatus were protected from atmospheric moisture with a nitrogenblanket. Toluene (100 mL) was added and the contents of the apparatusrefluxed until no further evolution of water occurred. The apparatus wasdisassembled and the toluene was decanted before the flask was connectedto a vacuum line to strip off toluene entrapped in the reaction mass.The product was used without further purification. The FTIR spectrum ofthe reaction product shows strong carbonyl adsorptions at 1736 cm⁻¹ and1672 cm⁻¹. The reaction product is estimated to be an approximately 4:1mixture of 2-aminoethyl glycinate and N,O-diglycyl ethanolamine bycomparison with the spectra of ethyl glycinate hydrochloride and glycylglycine hydrochloride.

Cholinyl Lysinate

Concentrated sulfuric acid (11.40 g, 0.120 mole) was diluted into water(10 mL). Lysine monohydrochloride (13.69 g, 0.075 mole), cholinechloride (10.47 g, 0.075 mole), and the diluted sulfuric acid was placedinto a 250 mL 1-neck round bottom flask fitted with a magnetic stirringbar, heated in a thermostatted oil bath, and connected to a vacuum line.Vacuum was gradually applied to the flask and then heat graduallyincreased in order to remove volatiles into a trap cooled in liquidnitrogen. The reaction was terminated when the bath temperature reached110° C. and the pressure decreased to 0.024 mm-Hg. The product ishomogeneous by thin layer chromatography (cellulose, aceticacid/acetonitrile/water 5:65:30 v/v/v, developed with ninhydrin spray,Rf=0.25). The product was used directly.

1,3-Propanediyl Diglycinate

Concentrated sulfuric acid (10.78 g, 0.110 mole) was diluted into water(10 mL). 1,3-Propanediol (7.61 g, 0.100 mole), glycine (15.0 g, 0.200mole), and the diluted sulfuric acid were placed in a 250 mL, 3-neck,round bottom flask fitted with a stopper, a mechanical stirrer, athermostatted oil bath, and a Dean-Stark water trap. The contents of theapparatus were protected from atmospheric moisture with a nitrogenblanket. Toluene (100 mL) was added, the oil bath thermostatted at 130°C., and the contents of the apparatus refluxed until no furtherevolution of water occurred (ca. 9 hours). The apparatus wasdisassembled and the toluene decanted. The reaction mass was dissolvedin water (29 mL) by stirring at room temperature. Upon cooling to −20°C. for 18 hours, the solution deposits fine white crystals which areremoved by filtration. The filtrate is poured into methanol (250 mL),precooled to 3° C., to deposit a semi-solid paste. The supernatant wasdecanted, and the paste triturated in several portions in a mortar andpestle with methanol (50 mL) to yield a granular solid (12.55 g). Asample of solid (9.19 g) was boiled with methanol (18.4 mL) plus water(7.9 mL), filtered while hot, and allowed to crystallize at 4° C. for 18hours. The precipitate was filtered while cold, compacted on the funnelunder a dam, rinsed with methanol, acetone, and air dried, to yield awhite crystalline solid (6.89 g). A sample of this material was titratedwith aqueous KOH using a pH meter. The apparent equivalent weight peramine is 201 g/mole; an acidic contaminant with an apparent equivalentweight of 601 g/mole was also present. The FTIR shows a single carbonylabsorption at 1744 cm⁻¹.

16. Fermentation Conditions

The fermentors used for the expression of protein polymers were usuallya 15 L MBR, 10 L working volume, or a 13 L Braun Biostat E, 8.5 Lworking volume. The choice of the fermentor and its size is notcritical. Any media used for the growth of E. coli can be used. Thenitrogen source ranged from NZAmine to inorganic salts and the carbonsource generally used was glycerol or glucose. All fermentations weredone with the appropriate selection conditions imposed by the plasmidrequirements (e.g. kanamycin, ampicillin, etc.). The fermentation methodused to express protein polymers in E. coli was the fed-batch method.This is the preferred method for the fermentation of recombinantorganisms even if other methods can be used.

The fed-batch method exploits the stage of cell growth where theorganisms make a transition from exponential to stationary phase. Thistransition is often the result of either depletion of an essentialnutrient or accumulation of a metabolic byproduct. When the transitionis the result of nutrient depletion, the addition of nutrients to thesystem causes cell division to continue. One or more essential nutrientscan incrementally be added to the fermentation vessel during the run,with the net volume increasing during the fermentation process. Theresult is a controlled growth rate where biomass and expression levelscan be optimized. When the cell number in the culture has reached or isapproaching a maximum, protein polymer production is induced byproviding an appropriate physical or chemical signal, depending upon theexpression system used. Production will then continue until theaccumulated product reaches maximum levels (Fiestchko, J., and Ritch,T., Chem. Eng. Commun. 1986, 45:229-240; Seo, J. H.; Bailey, J. E.,Biotechnol. Bioeng. 1986, 28:1590-1594).

Example 2

Construction of SELP8K, SELP8E and CLP6

Polymers were prepared designated SELP8K and SELP8E, which arecharacterized by having functional groups for cross-linking. Theconstruction of these polymers is described below starting from theprevious gene monomer, SELP0 (see U.S. Pat. No. 5,243,038, pSY1298).

SELP8K and SELP8E Amino Acid Monomer Sequence Design

SELP8K MONOMER (GAGAGS)₄ (GVGVP)₄ GKGVP (GVGVP)₃ (SEQ ID NO:04)

SELP8E MONOMER (GAGAGS)₄ (GVGVP)₄ GEGVP (GVGVP)₃ (SEQ ID NO:05)

SELP8 Construction

Plasmid pSY1378 (see U.S. Pat. No. 5,243,038) was digested with BanIREN, purified using agarose gel electrophoresis followed by NACS column,and the DNA was then ethanol precipitated in 2.5 M ammonium acetate andligated with pPT0134 (See PCT\US92\09485) previously digested with FokIREN, phenol/chloroform extracted and ethanol precipitated.

The products of the ligation mixture were transformed into E. colistrain HB101. Plasmid DNA from transformants was purified and analyzedby digestion using NruI and XmnI RENs. Plasmid pPT0255 containing thedesired restriction pattern was obtained and was used for subsequentconstructions.

Plasmid DNA pPT0255 was treated with Cfr10I REN followed by RNAse. Thedigestion fragments were separated by agarose gel electrophoresis, theDNA was excised and self-ligated. The products of the ligation mixturewere transformed into E. coli strain HB101. Plasmid DNA fromtransformants was purified and analyzed by digestion using NaeI and StuIRENs. Plasmid pPT0267 containing the desired deletion was used forsubsequent constructions.

Two oligonucleotide strands as shown in Table 1 were synthesized andpurified as described in Example 1.

TABLE 1 (SEQ ID NO:06) 5′-CTGGAGCGGGTGCCTGCATGTACATCCGAGT-3′ (SEQ IDNO:07) 3′-CCGAGACCTCGCCCACGGACGTACATGTAGGCTCA-5′

The two oligonucleotide strands were annealed and ligated with the DNAof plasmid pPT0267 which had been previously digested with BanII andScaI RENs, and purified by agarose gel electrophoresis followed by NACScolumn.

The products of this ligation reaction were transformed into E. colistrain HB101. Plasmid DNA from transformants was purified and digestedwith DraI. Plasmid DNA from two clones that gave the correct digestionpattern was sequenced. One plasmid DNA, designated pPT0287, was found tobe correct and chosen for further constructions.

Plasmid DNA pSY1298 (see U.S. Pat. No. 5,243,038) was digested withBanII REN, and the SELP0 gene fragment was purified by agarose gelelectrophoresis followed by NACS and then ligated to pPT0287 digestedwith BanII. The enzyme was then removed using phenol/chloroformextraction and ethanol precipitation.

The products of the ligation mixture were transformed into E. colistrain HB101. Plasmid DNA from transformants was purified and analyzedby digestion using DraI REN. Plasmid DNA from the clones showing thecorrect restriction pattern was further digested with BanII, AhaII andStuI RENs. Plasmid pPT0289 contained the desired SELP8 monomer sequence(see Table 2).

TABLE 2 SELP8 Gene Monomer Sequence BanI                          BanIIGGT GCC GGT TCT GGA GCT GGC GCG GGC TCT GGA GTA GGT GTG CCA GGT CCA CGGCCA AGA CCT CGA CCG CGC CCG AGA CCT CAT CCA CAC GGT CCAG   A   G   S   G   A   G   A   G   S   G   V   G   V   P   G GTA GGAGTT CCG GGT GTA GGC GTT CCG GGA GTT GGT GTA CCT GGA GTG CAT CCT CAA GGCCCA CAT CCG CAA GGC CCT CAA CCA CAT GGA CCT CACV   G   V   P   G   V   G   V   P   G   V   G   V   P   G   V                              SmaI GGT GTT CCA GGC GTA GGT GTG CCC GGGGTA GGA GTA CCA GGG GTA GGC CCA CAA GGT CCG CAT CCA CAC GGG CCC CAT CCTCAT GGT CCC CAT CCGG   V   P   G   V   G   V   P   G   V   G   V   P   G   V   G                                             BanII GTC CCT GGA GCG GGTGCT GGT AGC GGC GCA GGC GCG GGC TCT GGA GCG CAG GGA CCT CGC CCA CGA CCATCG CCG CGT CCG CGC CCG AGA CCT CGCV   P   G   A   G   A   G   S   G   A   G   A   G   S   G   A (SEQ IDNOS: 08 & 09)

Construction of SELP8K and SELP8E Gene Monomers

One oligonucleotide strand coding for a portion of the SELP8 genemonomer was synthesized with a single base polymorphism at position 90.The use of both adenine and guanidine at this position producedoligonucleotides from a single synthesis that encoded the amino acidslysine and glutamic acid (see Table 3). The synthesis was conductedusing an Applied Biosystems DNA synthesizer model 381A and a 2000 Åsynthesis column supplied by Glen Research. During the synthesis therequired interrupt-pauses for bottle changes were minimized. After thesynthesis the 202 base DNA fragment was deprotected and cleaved from thecolumn support by treatment in 30% ammonium hydroxide at 55° C. for 6hours.

TABLE 35′-ATGGCAGCGAAAGGGGACCGGGCTCTGGTGTTGGAGTGCCAGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGA(A/G)AAGGTGTTCCGGGGGTAGGTGTGCCGGGCGTTGGAGTACCAGGTGTAGGCGTCCCGGGAGCGGGTGCTGGTAGCGGCGCAGGCGCGGGCTCTTTCCGCTAAAGTCCTGCCGT-3′(SEQ ID NO:10)

Two additional DNA strands were used as primers for PCR amplification.

The two strands were

1. 5′-AAGAAGGAGATATCATATGGCAGCGAAAGGGGACC-3′ (SEQ ID NO:11)

2. 5′-CGCAGATCTTTAAATTACGGCAGGACTTTAGCGGAAA-3′ (SEQ ID NO:12)

The PCR reaction was carried out and the reaction product was purifiedas described in Example 1.

The DNA was resuspended and digested with BanII REN as described inExample 1. The digested DNA was then separated by low-melting agarosegel electrophoresis and ligated with pPT0289 previously digested withBanII RENs and purified by NACS column. The products of the ligationreaction were transformed into E. coli strain HB101. Plasmid DNA fromisolated transformants was purified and analyzed by digestion usingApaLI and EcoNI RENs. Plasmid DNA from the clones showing the correctrestriction pattern were further analyzed by digestion using Asp700 RENto distinguish between clones encoding a lysine or glutamic acid at thepolymorphic position. Plasmid DNA from clones containing each of thepolymorphs was purified and analyzed by DNA sequencing. Plasmid pPT0340contained the desired SELP8K monomer sequence (see Table 4) and pPT0350contained the desired SELP8E monomer sequence.

TABLE 4 SELP8K Gene Monomer Sequence BanI                          BanIIGGT GCC GGT TCT GGA GCT GGC GCG GGC TCT GGT GTT GGA GTG CCA GGT CCA CGGCCA AGA CCT CGA CCG CGC CCG AGA CCA CAA CCT CAC GGT CCAG   A   G   S   G   A   G   A   G   S   G   V   G   V   P   G                                              EcoNI GTC GGT GTT CCG GGTGTA GGC GTT CCG GGA GTT GGT GTA CCT GGA AAA CAG CCA CAA GGC CCA CAT CCGCAA GGC CCT CAA CCA CAT GGA CCT TTTV   G   V   P   G   V   G   V   P   G   V   G   V   P   G   K GGT GTTCCG GGG GTA GGT GTG CCG GGC GTT GGA GTA CCA GGT GTA GGC CCA CAA GGC CCCCAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CAT CCGG   V   P   G   V   G   V   P   G   V   G   V   P   G   V   G    SmaI                                         BanII GTC CCG GGA GCGGGT GCT GGT AGC GGC GCA GGC GCG GGC TCT GGA GCG CAG GGC CCT CGC CCA CGACCA TCG CCG CGT CCG CGC CCG AGA CCT CGCV   P   G   A   G   A   G   S   G   A   G   A   G   S   G   A (SEQ IDNO:13 & 14)

SELP8K Polymer Construction

Plasmid DNA from pPT0340 was digested with BanI REN and the digestionfragments were separated by agarose gel electrophoresis. The SELP8K genefragment, 192 bp, was excised and purified by NACS column. The purifiedfragment was ligated with plasmid pPT0317 which had been digested withBanI REN, passed through a Millipore Probind and a Bio-Spin 6 column.The DNA was then treated with shrimp alkaline phosphatase (SAP) asdescribed in Example 1.

The products of this ligation reaction were transformed into E. colistrain HB101. Transformants were selected for resistance to kanamycin.Plasmid DNA from individual transformants was purified and analyzed forincrease size due to SELP8K monomer multiple DNA insertion. Severalclones were obtained with insert sizes ranging from 200 bp toapproximately 7 kb. Clones containing from 6 to 32 repeats, were usedfor expression of the SELP8K protein polymer (pPT0341, pPT0343, pPT0344,pPT0345 and pPT0347).

SELP8K Expression Analysis

An overnight culture which had been grown at 30° C. was used toinoculate 50 ml of LB media contained in a 250 ml flask. Kanamycin wasadded at a final concentration of 50 μg per ml and the culture wasincubated with agitation (200 rpm) at 30° C. When the culture reached anOD₆₀₀ of 0.8, 40 ml were transferred to a new flask prewarmed at 42° C.and incubated at the same temperature for approximately 2 hours. Thecultures (30° and 42°) were chilled on ice and OD₆₀₀ was taken. Cellswere collected by centrifugation and divided in 1.0 OD₆₀₀ aliquots andused to perform western analysis using anti-SLP antibody.

E. coli strain HB101 containing plasmids pPT0341, pPT0343, pPT0344,pPT0345 and pPT0347 were grown as described above. The proteins producedby these cells were analyzed by Western blot for detection proteinsreactive to SLP antibodies. Each clone produced a strongly reactiveband. The apparent molecular weights of the products ranged fromapproximately 35 kD to greater than 250 kD. Strain pPT0345 produced anSLP antibody reactive band of apparent molecular weight 80,000. Theexpected amino acid sequence of the SELP8K polymer encoded by plasmidpPT0345 is shown below.

pPT0345            SELP8K      884 AA            MW 69,772      MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPMGAGSGAGAGS       [(GVGVP)₄ GKGVP(GVGVP)₃ (GAGAGS)₄ ]₁₂       (GVGVP)₄ GKGVP (GVGVP)₃ (GAGAGS)₂       GAGAMDPGRYQDLRSHHHHHH (SEQ ID NO:15)

SELP8K Purification

SELP8K was produced in E. coli strain pPT0345 by fermentation. Theproduct was purified from the cellular biomass by means of cellularlysis, clearance of insoluble debris by centrifugation, and affinitychromatography. The purified product was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis, immunoreactivity with apolyclonal antisera which reacts with silk-like peptide blocks (SLPantibody), and amino acid analysis. A protein band of apparent molecularweight 80,000 was observed by amido black staining of SDS-PAGE separatedand transferred samples and the same band reacted with the SLP antibodyon Western blots. As expected, amino acid analysis (shown in Table 5)indicated that the product was enriched for the amino acids glycine(43.7%), alanine (12.3%), serine (5.3%), proline (11.7%), and valine(21.2%). The product also contained 1.5% lysine. The amino acidcomposition table below shows the correlation between the composition ofthe purified product and the expected theoretical compositions asdeduced from the synthetic gene sequence.

TABLE 5 Amino Acid Analysis of Purified SELP8K Theoretical % Amino Acidpmoles Actual % composition composition Ala 1623.14 12.3 12.2 Asx 122.200.9 0.8 Glx nd nd 0.4 Phe 58.16 0.4 0.1 Gly 5759.31 43.7 41.5 His 46.750.4 0.8 Ile 43.87 0.3 0 Lys 198.21 1.5 1.5 Leu 39.54 0.3 0.5 Met 36.010.3 0.3 Pro 1534.21 11.7 12.4 Arg 70.84 0.5 0.6 Ser 703.83 5.3 6.1 Thrnd nd 0.1 Val 2797.47 21.2 22.4 Tyr 140.87 1.1 0.1 nd = none detected

CLP6 Preparation

CLP6 was prepared as described in PCT/US92/09485 using strain pPT0246(CLP6 referred to as DCP6). The protein polymer was purified inmultigram quantities using standard protein purification, extraction,and separation methods. The lyophilized product was a white, spongymaterial, extremely soluble in water.

CLP6 pPTO246        1,065 AA       MW 85,386MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM [(GAHGPAGPK)₂ (GAQGPAGPG)₂₄(GAHGPAGPK)₂]₄ GAMDPGRYQLSAGRYHYQLVWCCK (SEQ ID NO:16)

Example 3 The Construction of SELP0K Polymers

Polymer Design Elements

The copolymer structure of SELP8K consists of silk-like blocks (SLPblock) and elastin-like blocks (ELP block) in the following sequence:[(SLP block)₄ (ELP block)₈]. Additional polymers were designed to havedifferent resorption and solution properties by adjusting theirsilk-like to elastin-like block lengths while maintaining their adhesiveproperties. SELP0K contains half the length of crystallizable silk-likeblocks than SELP8K while maintaining the dispersion frequency withrespect to the elastin-like segments.

Polymers with intervening sequences to promote in vivo resorptionthrough proteolytic cleavage by collagenase (92 kd) and cathepsins werealso designed. SELP0K is used as the backbone for these designs, butthese sites can be used in many different polymer backbone sequences.The insert location is chosen to permit accessibility of the site to thecatalytic groove of the protease. Most proteases will bind up to 4upstream amino acids from the cleavage site. Therefore, the insertsequences should be free of hydrogen bonding and crystallization thatmay be induced by, for example, silk-like blocks.

The beta structure of the SELP0K will break after the proline of thefirst elastin-like block. SELP0K-CS1 contains two adjacent cleavagesites for collagenase (PLGP) (SEQ ID NO: 17) within a six amino acidinsert. The insertion site was chosen to be removed from the silk-likeblocks by at least one proline amino acid (GAGAGS GVGVP L G P L G PGVGVP) (SEQ ID NO: 18). SELP0K-CS2 contains multiple cleavage sites forcathepsins B (ARR), L (FF), S and H (FVR) and plasmin (R) within aneight amino acid insert. The insertion site was chosen to be removedfrom the silk-like blocks by at least one proline amino acid (GAGAGSGVGVP G F F V R A R R GVGVP)(SEQ ID NO: 19).

Construction of Plasmid pPT0317

Plasmid DNA pSY1262 (see U.S. Pat. No. 5,243,038) was linearized withPvuII REN, then passed through a Probind filter and a Bio-Spin 6 column.The DNA was then treated with Shrimp Alkaline Phosphatase (SAP). Thelinearized pSY1262 DNA was then ligated with a DNA fragment from pQE-17(QIAGEN Catalog #33173) prepared as follows. Plasmid DNA pQE-17 wasdigested with BgIII and HindIII RENs and the 36 bp fragment shown inTable 6 was purified using a Probind filter and a Biospin column. TheDNA was purified further using a Microcon-30 filter and the filtrate,containing the 36 bp fragment, was kept. The DNA was then treated withDNA Polymerase I and purified using a Probind filter and a Biospincolumn (see Example 1).

TABLE 6 (SEQ ID NO:20) 5′-GATCTTCGATCTCATCACCATCACCATCACTA (SEQ IDNO:21)     3′-AAGCTAGAGTAGTGGTAGTGGTAGTGATTCGT

The product of the ligation reaction was transformed into E. coli strainHB101. Plasmid DNA from transformants was purified and analyzed bydigestion using Bst1107I and EcoRV RENs. The clones containing thedesired DNA fragment were further digested with Bst1107I and BstYI RENsto determine the orientation of the insert. Plasmid DNA from the clonesshowing the correct restriction pattern was purified and analyzed by DNAsequencing. Plasmid pPT0317 contained the desired DNA insert and wasused for further DNA constructions.

SELP0K Polymer Construction

One oligonucleotide strand as shown in Table 7 was synthesized using anApplied Biosystems DNA synthesizer model 381A and a 2000 Å synthesiscolumn supplied by Glen Research. After the synthesis the 93 base DNAfragment was deprotected and cleaved from the column support bytreatment in ammonium hydroxide at 55° C. for 6 hours.

TABLE 7 5′-ATGGCAGCGAAAGGGGACCGGTGCCGGCGCAGGTAGCGGAGCCGGTGCGGGCTCAAAAAGGGCTCTGGTGCCTTTCCGCTAAAGTCCTGCCGT -3′ (SEQ ID NO:22)

The PCR reaction was performed using the same two DNA primer strands asdescribed for the construction of the SELP8K gene monomer and thereaction product was purified. The DNA was resuspended and digested withBanI REN. The digested DNA was then separated by low-melting agarose geland ligated with pPT0285 (see PCT/US92/09485) previously digested withBanI REN and purified by NACS column. The product of the ligationreaction was transformed into E. coli strain HB101. Plasmid DNA fromtransformants was purified and analyzed by digestion using EcoRI andBanII RENs. Plasmid DNA from the clones showing the correct restrictionpattern was then purified and analyzed by DNA sequencing. PlasmidpPT0358 contained the desired sequence and was used for subsequent DNAconstructions.

Plasmid DNA from pPT0340 was digested with BanII REN and the digestionfragments were separated by agarose gel electrophoresis. The SELP0K genefragment, 156 bp, (see Table 8), was excised and purified using anUltrafree-MC filter followed by Bio-Spin 6 column.

TABLE 8 BanII G GGC TCT GGT GTT GGA GTG CCA GGT GTC GGT GTT CCG GGT GTAGGC GTT C CCG AGA CCA CAA CCT CAC GGT CCA CAG CCA CAA GGC CCA CAT CCGCAA   G   S   G   V   G   V   P   G   V   G   V   P   G   V   G   V CCGGGA GTT GGT GTA CCT GGA AAA GGT GTT CCG GGG GTA GGT GTG CCG GGC CCT CAACCA CAT GGA CCT TTT CCA CAA GGC CCC CAT CCA CAC GGCP   G   V   G   V   P   G   K   G   V   P   G   V   G   V   P GGC GTTGGA GTA CCA GGT GTA GGC GTC CCG GGA GCG GGT GCT GGT AGC CCG CAA CCT CATGGT CCA CAT CCG CAG GGC CCT CGC CCA CGA CCA TCGG   V   G   V   P   G   V   G   V   P   G   A   G   A   G   S               BanII GGC GCA GGC GCG GGC TC CCG CGT CCG CGC CCG AGG   A   G   A   G   S (SEQ ID NOS:23 & 24)

The purified fragment was ligated with plasmid pPT0358 which had beendigested with BanII REN, then passed through a Probind filter and aMicrocon-30 filter. The digestion fragments were then separated byagarose gel electrophoresis. The plasmid DNA was then excised andpurified using an Ultrafree-MC filter followed by Bio-Spin 6 column (seeExample 1).

The product of this ligation reaction was transformed into E. colistrain HB101. Transformants were selected for resistance tochloramphenicol. Plasmid DNA from individual transformants was purifiedand analyzed for increased size due to SELP0K multiple DNA insertion.Several clones were obtained with inserts of different sizes. PlasmidpPT0359, pPT0360 and pPT0374 containing respectively 18, 2 and 6 repeatsof the SELP0K gene monomer were used for subsequent constructions.

Plasmid DNA from pPT0359 and pPT0374 was digested with BanI REN and thedigestion fragments were separated by agarose gel electrophoresis. TheSELP0K gene fragments, approximately 2800 bp and 1000 bp, were excisedand purified by NACS column. The purified fragments were then ligatedwith plasmid pPT0317 which had been digested with BanI REN, then passedthrough a Probind filter and a Bio-Spin 6 column. The DNA was thentreated with Shrimp Alkaline Phosphatase (SAP), passed through a Probindfilter and then a Bio-Spin 6 column (see Example 1).

The product of these ligation reactions was transformed into E. colistrain HB101. Transformants were selected for resistance to kanamycin.Plasmid DNA from individual transformants was purified and analyzed forincreased size due to SELP0K multiple DNA insertion. Several clones wereobtained. Plasmid pPT0364 and pPT0375 were chosen to be used forexpression of SELP0K.

SELP0K Expression Analysis

E. coli strain HB101 containing plasmid pPT0364 and pPT0375 were grownas described in Example 1. The proteins produced by these cells wereanalysed by SDS-PAGE for detection of reactivity to ELP antibodies. Inevery analysis a strong reactive band was observed of an apparentmolecular weight of approximately 95 kD and 35 kD respectively.

pPTO364       SELPOK      1000 AA   MW 80,684    MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM     [(GAGAGS)₂ (GVGVP)₄GKGVP(GVGVP)₃]₁₈     (GAGAGS)₂ GAGAMDPGRYQDLRSHHHHHH     (SEQ ID NO:25)pPTO375       SELPOK      376 AA   MW 31,445    MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM     [(GAGAGS)₂ (GVGVP)₁ GKGVP(GVGVP)₃]₆     (GAGAGS)₂GAGAMDPGRYQDLRSHHHHHH     (SEQ ID NO:26)

SELP0K-CS1 Polymer Construction

Plasmid pPT0360 was digested with BanI REN and the digestion fragmentswere separated by agarose gel electrophoresis. The SELP0K gene fragment,approximately 300 bp, was excised and purified using an Ultrafree-MCfilter followed by Bio-Spin 6 column. The purified fragment was ligatedwith plasmid pPT0134 (see PCT/US92/09485) which had been digested withFold REN. The enzyme was heat inactivated at 65° C. for 20 minutes andthe ligation mixture was then passed through a Probind filter. The DNAwas then treated with Shrimp Alkaline Phosphatase (SAP), passed througha Probind filter and then a Bio-Spin 6 column.

The product of this ligation reaction was transformed into E. colistrain HB101. Transformants were selected for resistance tochloramphenicol. Plasmid DNA from individual transformants was purifiedand analyzed by digestion using DraI REN. One plasmid, pPT0363, showedthe correct restriction pattern and was used for subsequent DNAconstructions.

One oligonucleotide strand as shown in Table 9 was synthesized using anApplied Biosystems DNA synthesizer model 381A and a 2000 Å synthesiscolumn supplied by Glen Research. After the synthesis the 141 base DNAfragment was deprotected and cleaved from the column support bytreatment in ammonium hydroxide at 55° C. for 6 hours.

TABLE 95′-ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGCCGCTGGGTCCTCTTGG (SEQID NO:27)CCCAGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT-3′

The PCR reaction was performed using the same two DNA primer strands asdescribed for the construction of the SELP8K gene monomer and thereaction product was purified. The DNA was then resuspended and digestedwith BsrFI and EcoNI RENs. The digested DNA was treated with Probind andMicrocon-30 filters, a Bio-Spin 6 column, and then ligated with pPT0363previously digested with BsrFI REN, treated with a ProBind filter and aBio-Spin 6 column and then further digested with EcoNI REN. Thedigestion fragments were separated by agarose gel electrophoresis. Thelarger DNA band, approximately 2000 bp, was excised and purified usingan Ultrafree-MC filter followed by Bio-Spin 6 column (see Example 1).

The product of the ligation reaction was transformed into E. coli strainHB101. Plasmid DNA from individual transformants was purified andanalyzed by digestion using Asp700I and EcoO109I RENs. Plasmid DNA fromthe clones showing the correct restriction pattern was then purified andanalyzed by DNA sequencing. Plasmid pPT0368 (see Table 10) contained thedesired sequence and was used for subsequent DNA constructions.

TABLE 10 BanII (SEQ ID NOS: 28 & 29) G GGC TCT GGT GTT GGA GTG CCG CTGGGT CCT CTT GGC CCA GGT GTC C CCG AGA CCA CAA CCT CAC GGC GAC CCA GGAGAA CCG GGT CCA CAG  G   S   G   V   G   V   P   L   G   P   L   G   P   G   V GGT GTT CCGGGT GTA GGC GTT CCG GGA GTT GGT GTA CCT GGA AAA CCA CAA GGC CCA CAT CCGCAA GGC CCT CAA CCA CAT GGA CCT TTTG   V   P   G   V   G   V   P   G   V   G   V   P   G   K GGT GTT CCGGGG GTA GGT GTG CCG GGG GTT GGA GTA CCA GGT GTA CCA CAA GGC CCC CAT CCACAC GGC CCG CAA CCT CAT GGT CCA CATG   V   P   G   V   G   V   P   G   V   G   V   P   G   V                                                   BanII GGC GTC CCG GGAGCG GGT GCT GGT AGC GGC GCA GGC GCG GGC TCT CCG CAG GGC CCT CGC CCA CGACCA TCG CCG CGT CCG CGC CCG AGAG   V   P   G   A   G   A   G   S   G   A   G   A   G   S

Plasmid DNA pPT0368 was digested with BanII REN, and the digestionfragments were separeted by agarose gel electrophoresis. The SELP0K-CS1gene fragment, 174 bp, was excised and purified using an Ultrafree-MCfilter followed by Bio-Spin 6 column. The purified fragment was ligatedwith plasmid pPT0358 which had been digested with BanII REN, then passedthrough a Probind filter and a Microcon-30 filter. Subsequently thedigestion fragments were separated by agarose gel electrophoresis. Theplasmid DNA was then excised and purified using an Ultrafree-MC filterfollowed by Bio-Spin 6 column (see Example 1).

The product of this ligation reaction was transformed into E. colistrain HB101. Transformants were selected for resistance tochloramphenicol. Plasmid DNA from individual transformants was purifiedand analyzed for increased size due to SELP0K-CS1 multiple DNAinsertion. Several clones were obtained with insert sizes ranging from1000 bp to approximately 3000 bp. Plasmid pPT0369 containing 16 repeatsof the SELP0K-CS1 gene monomer was used for subsequent constructions.

Plasmid DNA from pPT0369 was digested with BanI REN, followed by aProbind filter and then the digestion fragments were separated byagarose gel electrophoresis. The SELP0K-CS1 gene fragment, approximately2800 bp, was excised and purified by an Ultrafree-MC filter and desaltedusing a Bio-Spin 6 column. The purified fragments were then ligated withplasmid pPT0317 which had been digested with BanI REN and then passedthrough a Probind filter and a Bio-Spin 6 column. The DNA was thentreated with Shrimp Alkaline Phosphatase (SAP), passed through a Probindfilter and then a Bio-Spin 6 column (see Example 1).

The product of these ligation reactions was transformed into E. colistrain HB101. Transformants were selected for resistance to kanamycin.Plasmid DNA from individual transformants was purified and analyzed forincreased size due to SELP0K-CS1 multiple DNA insertion. Several cloneswere obtained. Plasmid pPT0370 was chosen to be used for expression ofSELP0K-CS 1.

SELP0K-CS1 Expression Analysis

E. coli strain HB101 containing plasmid pPT0370 was grown as describedin Example 1. The proteins produced by these cells were analysed bySDS-PAGE for detection of reactivity to ELP antibodies. In everyanalysis a strong reactive band was observed with an apparent molecularweight of approximately 90 kD.

pPT0370           SELPOK-CS1        934 AA          MW 76,389 (SEQ IDNO:30)       MDPVVLQRRDWENPGVTQLNRLAAHPPFASDPM       [(GAGAGS)₂ (GVGVP)₁LGPLGP (GVGVP)₃ GKGVP (GVGVP)₃]₁₅       (GAGAGS)₂ GAGAMDPGRYQDLRSHHHHHH

SELP0K-CS2 Polymer Construction

One oligonucleotide strand as shown in Table 11 was synthesized using anApplied Biosystems DNA synthesizer model 381A and a 2000 Å synthesiscolumn supplied by Glen Research. After the synthesis the 147 base DNAfragment was deprotected and cleaved from the column support bytreatment in ammonium hydroxide at 55° C. for 6 hours.

TABLE 115′-ATGGCAGCGAAAGGGGACCGCCGGTGCGGGCTCTGGTGTTGGAGTGCCAGCCTTCTTTGTACGTG(SEQ ID NO:31)CACGCCGTGGTGTCGGTGTTCCGGGTGTAGGCGTTCCGGGAGTTGGTGTACCTGGAAAAGGTTTCCGCTAAAGTCCTGCCGT-3′

The PCR reaction was performed using the same two DNA primer strands asdescribed for the construction of the SELP8K gene monomer and thereaction product was purified. The DNA was then resuspended and digestedwith BsrFI and EcoNI RENs. The digested DNA was treated with ProBind andMicrocon-30 filters, a Bio-Spin 6 column, and then ligated with pPT0363previously digested with BsrFI REN, treated with a ProBind filter and aBio-Spin 6 column and then further digested with EcoNI REN. Thedigestion fragments were separated by agarose gel electrophoresis. Thelarger DNA band, approximately 2000 bp, was excised and purified usingan Ultrafree-MC filter followed by Bio-Spin 6 column.

The product of the ligation reaction was transformed into E. coli strainHB101. Plasmid DNA from individual transformants was purified andanalyzed by digestion using Asp700I and DraIII RENs. Plasmid DNA fromthe clones showing the correct restriction pattern was then purified andanalyzed by DNA sequencing. Plasmid pPT0367 (see Table 12) contained thedesired sequence and was used for subsequent DNA constructions.

TABLE 12 BanII (SEQ ID NOS: 32&33) G GGC TCT GGT GTT GGA GTG CCA GGC TTCTTT GTA CGT GCA CGC CGT C CCG AGA CCA CAA CCT CAC GGT CCG AAG AAA CATGCA CGT GCG GCA  G   S   G   V   G   V   P   G   F   F   V   R   A   R   R GGT GTC GGTGTT CCG GGT GTA GGC GTT CCG GGA GTT GGT GTA CCT GGA CCA CAG CCA CAA GGCCCA CAT CCG CAA GGC CCT CAA CCA CAT GGA CCTG   V   G   V   P   G   V   G   V   P   G   V   G   V   P   G AAA GGTGTT CCG GGG GTA GGT GTG CCQ GGC GTT GGA GTA CCA GGT GTA TTT CCA CAA GGCCCC CAT CCA CAC GGC CCG CAA CCT CAT GGT CCA CATK   G   V   P   G   V   G   V   P   G   V   G   V   P   G   V                                                   BanII GGC CTC CCC GGAGCG GGT GCT GGT AGC GGC GCA GGC GCG GGC TC CCG CAG GGC CCT CGC CCA CGACCA TCG CCG CGT CCG CGC CCG AGG   V   P   G   A   G   A   G   S   G   A   G   A   G   S

Plasmid DNA pPT0367 was digested with BanII REN, treated with a Probindfilter and a Bio-Spin6 column and then the digestion fragments wereseparated by agarose gel electrophoresis. The SELP0K-CS2 gene fragment,180 bp, was excised and purified using an Ultrafree-MC filter followedby Bio-Spin 6 column. The purified fragment was ligated with plasmidpPT0358 which had been digested with BanII REN and then passed through aProbind filter and a Microcon-30 filter. Subsequently the digestionfragments were separated by agarose gel electrophoresis. The plasmid DNAwas then excised and purified using an Ultrafree-MC filter followed byBio-Spin 6 column (see Example 1).

The product of this ligation reaction was transformed into E. colistrain HB101. Transformants were selected for resistance tochloramphenicol. Plasmid DNA from individual transformants was purifiedand analyzed for increased size due to SELP0K-CS2 multiple DNAinsertion. Several clones were obtained with insert sizes ranging from200 bp to approximately 3000 bp. Plasmid pPT0371 and pPT0372, containing18 and 15 repeats respectively of the SELP0K-CS2 gene monomer were usedfor subsequent constructions.

Plasmid DNA from pPT0372 was digested with BanI REN, followed by aProbind filter, and then the digestion fragments were separated byagarose gel electrophoresis. The SELP0K-CS2 gene fragment, approximately2800 bp, was excised and purified by an Ultrafree-MC filter and desaltedusing a Bio-Spin 6 column. The purified fragments were then ligated withplasmid pPT0317 which had been digested with BanI REN, passed through aProbind filter and then a Bio-Spin 6 column. The DNA was treated withShrimp Alkaline Phosphatase (SAP), passed through a Probind filter andthen a Bio-Spin 6 column (see Example 1).

The product of these ligation reactions was transformed into E. colistrain HB101. Transformants were selected for resistance to kanamycin.Plasmid DNA from individual transformants was purified and analyzed forincreased size due to SELP0K-CS2 multiple DNA insertion. Several cloneswere obtained. Plasmid pPT0373 was chosen to be used for the expressionof SELP0K-CS2.

SELP0K-CS2 Expression Analysis

E. coli strain HB101 containing plasmid pPT0373 was grown as describedin Example 1. The proteins produced by these cells were analysed bySDS-PAGE for detection of reactivity to ELP antibodies. In everyanalysis a strong reactive band was observed of an apparent molecularweight of approximately 90 kD.

pPT0373           SELPOK-CS2        964 AA          MW 83,218 (SEQ IDNO:34)       MDPWLQRRDWENPGVTQLNRLAAHPPFASDPM       [(GAGAGS)₂ (GVGVP)₁GFFVRARR (GVGVP)₃ GKGVP (GVGVP)₃)₁₅       (GAGAGS)₂GAGAMDPGRYQDLRSHHHHHH

SELP0K and SELP0K-CS1 Purification

SELP0K and SELP0K-CS 1 were produced in E. coli strains pPT0364 andpPT0370, respectively. The products were purified from the cellularbiomass by means of cellular lysis, clearance of insoluble debris bypolyethylene imine precipitation and centrifugation, ammonium sulfateprecipitation, and anion exchange chromatography. The purified productswere analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), immunoreactivity with a polyclonal antiserawhich reacts with elastin-like peptide blocks (ELP antibody), and aminoacid analysis.

For SELP0K, a protein band of apparent molecular weight 95,000 wasobserved by amido black staining of SDS-PAGE separated and transferredsamples and the same band reacted with the ELP antibody on Westernblots. As expected, amino acid analysis (shown in Table 13) indicatedthat the product was enriched for the amino acids glycine (41.0%),alanine (8.0%), serine (4.5%), proline (14.1%), and valine (26.8%). Theproduct also contained 1.9% lysine. The amino acid composition tablebelow shows the correlation between the composition of the purifiedproduct and the expected theoretical composition as deduced from thesynthetic gene sequence.

TABLE 13 Amino Acid Analysis of Purified SELP0K pMoles Mole %Theoretical Mole % ASX 28.10 0.6 0.7 GLX 26.90 0.6 0.4 SER 199.84 4.54.0 GLY 1812.07 41.0 40.5 HIS 28.45 0.6 0.7 ARG 20.49 0.5 0.5 THR 0 0.00.1 ALA 355.29 8.0 8.0 PRO 623.22 14.1 15.0 TYR 8.47 0.2 0.1 VAL 1183.6326.8 27.3 MET 7.21 0.4 0.3 ILE 4.83 0.1 0.0 LEU 20.66 0.5 0.4 PHE 7.570.2 0.1 LYS 84.02 1.9 1.8 Total 4420.75

For SELP0K-CS 1, a protein band of apparent molecular weight 90,000 wasobserved by amido black staining of SDS-PAGE separated and transferredsamples and the same band reacted with the ELP antibody on Westernblots. As expected, amino acid analysis (shown in Table 14) indicatedthat the product was enriched for the amino acids glycine (40.0%),alanine (7.6%), serine (5.2%), proline (16.3%), and valine (23.3%). Theproduct also contained 1.5% lysine. The amino acid composition tablebelow shows the correlation between the composition of the purifiedproduct and the expected theoretical composition as deduced from thesynthetic gene sequence.

TABLE 14 Amino Acid Analysis of Purified SELP0K-CS1 pMoles Mole %Theoretical Mole % ASX 16.43 0.7 0.7 GLX 10.59 0.5 0.4 SER 119.96 5.23.6 GLY 924.51 40.0 39.6 HIS 13.85 0.6 0.7 ARG 11.26 0.5 0.5 THR 0 0.00.1 ALA 175.07 7.6 7.3 PRO 376.40 16.3 16.7 TYR 2.49 0.1 0.1 VAL 537.9623.3 24.5 MET 5.19 0.2 0.3 ILE 0 0.0 0.0 LEU 76.62 3.3 0.4 PHE 2.58 0.10.1 LYS 35.68 1.5 1.6 Total 2308.59

Example 4 Evaluation of CLP6 and SELP8K Properties

Test Procedures

Tiseel Adhesive Systems. Rat skins were washed with water, blotted dryand cut into strips about 1 cm×4 cm. Adhesive from Tiseel Kit VH(Osterreiches Institute Fur Haemoderivate, GmbH, A-1220, Vienna,Austria) was applied according to the manufacturer's specifications.

Rat Skin Lap Shear Tensile Strength Assay. Adhesive formulations weretested for their ability to bond skin together using an in vitro ratskin lap shear tensile strength assay. Adhesives were applied to thesubcutaneous side of a strip of harvested rat skin. A second skin stripwas overlapped in order to produce an approximate bonding surface of 1cm². A 100 gram weight was applied to the lap joint and the adhesive wasallowed to cure, usually at room temperature for a period of 2 hours andwrapped in plastic to prevent desiccation. The lap joint was mounted onan Instron Tensile Tester or similar apparatus and tensile forceapplied. With the Instron, tensile force was typically applied at aconstant strain rate of 2 inches per minute. The load at failure wasrecorded and normalized to the measured area of overlap.

Adhesive Systems with Glutaraldehyde. Rat skins were washed with water,blotted dry, and cut into strips about 1 cm×4 cm. Glutaraldehyde wasdistilled, stored frozen and thawed immediately before use. Bovine serumalbumin was dissolved according to Goldman's specifications (Goldman,WO94/01508). CLP6 was dissolved at 600 mg/mL in 150 mM HEPES+30 mM NaCland adjusted to pH 7.5. SELP8K was dissolved at the concentrationsindicated in Table 15 in 150 mM HEPES+45 mM NaCl and adjusted to pH 8.The indicated aliquots of the solution of protein was spread over bothskins before the addition of the glutaraldehyde solution. The secondskin was overlaid, rubbed across the lower skin to distribute thecomponents, adjusted to an overlap area of ca. 1 cm², covered withplastic wrap to prevent drying, and cured for 2 hours at 25° C. under acompressive force of 100 g/cm².

Adhesive Systems with 1,6-(Diisocyanto)hexane. Rat skins were washedwith water, blotted dry, and cut into strips about 1 cm×4 cm. A solutionof SELP8K was made up in the specified buffer at a concentration of ca.50% w/w. A 1:1 v/v mixture of hexamethylene diisocyanate (HMDI) andPluronic L-61 surfactant was prepared. A 20 μL aliquot of SELP8Ksolution was applied to one skin followed by a 2 μL aliquot of thediluted HMDI. The second skin was overlaid, rubbed across the lower skinto mix the components, adjusted to ca. 1 cm² overlap, covered withplastic wrap to prevent drying, and cured for 2 hours at 25° C. under acompressive force of 100 g/cm².

Results

In order to provide a baseline for subsequent adhesive experiments,ethyl cyanoacrylate and Tiseel fibrin glue were evaluated. These resultsare reported in the following table.

TABLE 15 Base Case Lap Shear Tensile Strengths Tensile Strength ReagentDose g/cm² Normal Saline not applicable 13 ± 4 Tiseel Fibrin Glue ˜25mg   261 ± 51 Ethyl cyanoacrylate 25 mg  385 ± 119

All data reported are based on at least three test specimens. All testresults are based on a two hour cure time.

The subject compositions were compared to the proteinaceous adhesivesystem described by Goldman (WO94/01508). Ten microliters ofglutaraldehyde solution of the indicated concentration was added in allcases. The following table indicates the results.

TABLE 16 Lap Shear Tensile Strength of Glutaraldehyde Cured AdhesiveSystems Tensile Strength Reagent Dose g/cm² Ovalbumin + Glutaraldehyde 6mg/  50 ± 10 (30μ) 200 mg/mL 10 μL 2.5 N 2.5 mg Atelocollagen (denat) +Glutaraldehyde 3 mg/ 148 ± 47 (25 μL) 125 mg/mL 10 μL 2.5 N 2.5 mgCLP6 + Glutaraldehyde 24 mg/ 306 ± 98 (40 μL) 600 mg/mL 10 μL 2.5 N 2.5mg CLP6 + Glutaraldehyde 12 mg/ 171 ± 42 (20 μL) 600 mg/mL 10 μL 2.5 N2.5 mg SELP8K (30 μL) + Glutaraldehyde 18 mg/  545 ± 153 1 mg 600 mg/mL1.0 N 9 mg/ 452 ± 54 1 mg 300 mg/mL 1.0 N 9 mg/  234 ± 51* 1 mg 300mg/mL (impure) 1.0 N 9 mg/  210 ± 57* 0.1 mg 300 mg/mL (impure) 0.1 N 7mg/ 374 ± 90 2.5 mg 287 mg/mL 2.5 N 3 mg/ 361 ± 47 1 mg 100 mg/mL 1.0 N3 mg/ 274 ± 17 2.5 mg 100 mg/mL 2.5 N *This preparation of SELP8K wasknown to be impure and is estimated to yield adhesive strength aboutone-half of that of the more completely purified material.

The data in the above table demonstrate that the subject polymers areable to provide superior adhesive capabilities when used in theglutaraldehyde cured system under conditions comparable to collagen andovalbumin. Despite the lower number of amino groups available forcrosslinking, the SELP8K polymer provides the highest tensile strengthsin the rat skin lap shear results. The above results demonstrate thatsignificant adhesion can be obtained at even low doses of glutaraldehydedown to 100 μg/cm². The quality and purity of the glutaraldehyde isknown to be critical to obtain good crosslinking (Rujigrok, DeWijn,Boon, J. Matr. Sci. Matr. Med. 5:80-87 (1994); Whipple, Ruta, J. Org.Chem. 39:1666-1668 (1974). The glutaraldehyde used in these experimentswas distilled, diluted to 2.5N and stored at −20° C. until used.

In the next study, hexamethylene diisocyanate was employed. It was foundnecessary to add an equal volume of diluent to obtain good adhesion,since the curing was otherwise too fast. The following table indicatesthe results, where n=12.

TABLE 17 Lap Shear Tensile Strength of HMDI Derived Adhesive SystemTensile Strength Reagent Dose g/cm² SELP8K       20 μL × 50% w/w 10 mg 585 ± 203 HMDI/L-61 1:1 v/v  2 μL × 50% v/v  1 mg Buffer: (100 μLwater + 10 μL 1 M KHCO₃) SELP8K       20 μL × 50% w/w 10 mg 503 ± 21HMDI/L-61 1:1 v/v  2 μL × 50% v/v  1 mg Buffer: (100 μL 50 mM PO₄ (pH6.8) + 5 μL 1 M KHCO₃) SELP8K       20 μL × 50% w/w 10 mg 451 ± 67HMDI/L-61 1:1 v/v  2 μL × 50% v/v  1 mg Buffer: (100 μL 50 mM PO₄ (pH6.8) + 10 μL 1 M KHCO₃) SELP8K       20 μL × 50% w/w 10 mg 362 ± 71HMDI/L-61 1:1 v/v  2 μL × 50% v/v  1 mg Buffer: (100 μL 50 mM PO₄ (pH6.8))

Example 5 Evaluation of SELP0K (SE0K) AND SELP0K-CS 1 Properties

A number of formulations were prepared using different components forthe formulation and determining the lap shear strength. In addition avariety of protocols were used to prepare the protein dope to provideadhesion. These protocols are set forth as follows:

Protocol A. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 13.1:1

Preparation of Protein Dope

The designation 1:2 refers to the nominal ratio of amino groups derivedfrom lysine to amino groups derived from SE0K. The designation 1:1refers to the nominal ratio of carbonate ions per amino group from SE0Kplus lysine. The designation 13.1:1 refers to the nominal ratio ofisocyanate groups to amine groups from SE0K plus lysine

A stock buffer solution was prepared by dissolving lysine hydrochloride0.0157 g, potassium carbonate 0.0710 g, and Evans Blue dye 0.00371 g in7.526 mL of deionized water. Stock buffer, 620.6 μL, was added to SE0K,127.1 mg in an Eppendorf tube. The mixture was agitated on a vortexmixer until complete dissolution occurred. The solution was centrifugedat about 5000 rpm for 30-60 seconds to separate air bubbles. Thesolution was then loaded into a 1 mL syringe for dispensing onto thetest skins. The optional inclusion of dye in the protein dope serves tomore readily visualize the distribution of the dope on the test skins.

Preparation of HMDI Setting Agent

The HMDI setting agent was prepared by dissolving Sudan Red dye, 1.75mg, in neat 1,6-diisocyanatohexane, 1.00 g. The optional inclusion ofdye in the setting agent serves to more readily visualize thedistribution of the setting agent on the test skins.

Preparation of Rat Skins

Freshly harvested rat hides were stored frozen at −20° C. Just beforeuse the hides were thawed and cut into 1 cm×3 cm strips. All fascia wasremoved from the strips of skin with a razor blade. Strips of skin wereselected which were uniform in width and thickness. Prepared rat skinsamples were temporarily stored at 37° C. between gauze pads soaked withPBS and contained in a plastic bag to prevent drying.

Application of Adhesive

In a 37° C. warm room, protein dope, 15 μL, was applied to each of twostrips of rat skin, 30 μL total, and aggressively worked into about a 1cm² area of each piece of skin with a stainless steel spatula. A totalof 1.8 μL of HMDI setting agent was applied to the skins, apportioned sothat 3 parallel stripes of HMDI were applied to the first skin and twostripes in an X-pattern were applied to the second skin. The skins wereimmediately assembled to form the lap joint, covered with a piece ofplastic film to prevent drying, and compressed under a 100 g weight. Thejoint was allowed to cure for 15 minutes at 37° C. The length and widthof the lap joint was measured to 1 mm using a ruler immediately beforetensile testing on an Instron Model 55 test machine. The crosshead speedwas set at 25 mm per minute. Lap shear tensile strengths were reportedin units of g/cm². Means and standard deviations were calculated formeasurements conducted at least in triplicate.

Protocol B. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 14.5:1

The steps of Protocol A were followed, except that bubbles were removedfrom the protein dope in a two stage process. After centrifugation, thedope was exposed to reduced pressure, 26 in-Hg, for 30 minutes. Thevolume of HMDI setting agent applied to the lap joint was 2.0 μL.

Protocol C. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 7.3:1

The steps of Protocol B were followed, except that the composition ofthe HMDI setting agent was altered. Sudan Red dye, 5.2 mg was dissolvedin 10.735 g of neat Pluronic surfactant L-31 by heating to 100° C. for10 minutes. After cooling to room temperature, an equal weight of1,6-diisocyanatohexane was added to this mixture. The mixture wasprepared immediately before use.

Protocol D1. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that Pluronic surfactantL-31, 4.57 mg, was added to SE0K, 74.6 mg. The ratio of SE0K to lysinebuffer solution remained as described in Protocol B.

Protocol D2. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that Pluronic surfactantL-31, 1.07 mg, was added to SE0K, 74.7 mg. The ratio of SE0K to lysinebuffer solution remained as described in Protocol B.

Protocol E. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 7.3:1

The steps of Protocol B were followed, except that in Protocol E, 5.1 mgPluronic L-31 was added to 85.0 mg SE0K. The composition of the HMDIsetting agent was also altered. Sudan Red dye, 5.2 mg was dissolved in10.735 g neat Pluronic surfactant L-31 by heating to 100° C. for 10minutes. After cooling to room temperature, an equal weight of1,6-diisocyanatohexane was added to this mixture. The mixture wasprepared immediately before use.

Protocol F. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that a stock buffersolution was prepared by dissolving lysine hydrochloride 0.46 g, boricacid 1.24 g in 92.2 mL of deionized water. The pH of this solution wasadjusted to pH 9.52 by the addition of 7.8 mL of 2 N sodium hydroxidesolution. Evans Blue dye, 0.50 mg/mL, was dissolved in this buffer, andthe solution filtered through a 0.45 micron syringe filter. Stockbuffer, 333.5 μL, was added to SE0K, 68.3 mg in an Eppendorf tube. Themixture was agitated on a vortex mixer until complete dissolutionoccurred.

Protocol G. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Borate pH9.5 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that a stock buffersolution was prepared by dissolving lysine hydrochloride, 0.46 g, andboric acid, 1.24 g in 99.1 mL of deionized water. The pH of thissolution was adjusted to pH 9.52 by the addition of 0.9 mL of 10 Npotassium hydroxide solution. Evans Blue dye, 0.50 mg/mL, was dissolvedin this buffer, and the solution filtered through a 0.45 micron syringefilter. Stock buffer, 367.2 μL, was added to SE0K, 75.2 mg in anEppendorf tube. The mixture was agitated on a vortex mixer untilcomplete dissolution occurred.

Protocol H. SE0K 17% w/w Lysine hydrochloride 1:2 Lithium Carbonate pH9.5 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving lysine hydrochloride, 42.7 mg, indeionized water, 10.0 mL, and adding lithium carbonate, 14.3 mg, to pH9.55. Evans Blue dye, 0.50 mg/mL, was dissolved in this buffer, and thesolution filtered through a 0.45 micron syringe filter.

Protocol I. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Carbonate pH9.5 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving sodium carbonate, 1.06 g, and lysinehydrochloride, 0.46 g, in 99.2 mL of deionized water. Using concentratedhydrochloric acid solution 0.8 mL, the solution was adjusted to pH 9.54.Evans Blue dye, 0.50 mg/mL, was dissolved in this buffer, and thesolution filtered through a 0.45 micron syringe filter.

Protocol J. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate pH9.5 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving potassium carbonate, 1.38 g, andlysine hydrochloride, 0.46 g, in 99.1 mL of deionized water. Usingconcentrated hydrochloric acid solution, 0.9 mL, the solution wasadjusted to pH 9.53. Evans Blue dye, 0.50 mg/mL, was dissolved in thisbuffer, and the solution filtered through a 0.45 micron syringe filter.

Protocol K. SE0K 17% w/w Lysine hydrochloride 1:2 Cesium Carbonate pH9.5 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving lysine hydrochloride, 42.7 mg, indeionized water, 10.0 mL, and adding cesium carbonate, 55.2 mg, to pH9.52. Evans Blue dye, 0.50 mg/mL, was dissolved in this buffer, and thesolution filtered through a 0.45 micron syringe filter.

Protocol L. SE0K 17% w/w Lysine hydrochloride 1:2 Calcium Carbonate 1:1Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving lysine hydrochloride, 20.8 mg, indeionized water, 10.0 mL, and adding calcium carbonate, 68.6 mg. EvansBlue dye, 0.50 mg/mL, was dissolved in this buffer, and the solutionfiltered through a 0.45 micron syringe filter.

Protocol M. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate pH9.0 Isocyanate:Amine 14.5:1

The steps of Protocol B were followed, except that the stock buffersolution was prepared by dissolving lysine hydrochloride, 103.9 mg, indeionized water, 50.0 mL, and adding potassium carbonate, 473.3 mg.Using concentrated hydrochloric acid, this buffer was adjusted to pH9.00.

Protocol N1. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate1:1 Isocyanate:Amine 14.5:1 Iodide:Carbonate 1:2

The steps of Protocol B were followed, except that potassium iodide,5.70 mg/mL was added to the stock buffer solution. The nominal pH ofthis dope was about pH 11.

Protocol N2. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium CarbonatepH 9.0 Isocyanate:Amine 14.5:1 Iodide:Carbonate 1:2

The steps of Protocol B were followed, except that potassium iodide,5.70 mg/mL was added to the stock buffer solution. Using concentratedhydrochloric acid,this buffer was adjusted to pH 9.00.

Protocol O1. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate1:1 Isocyanate:Amine 14.5:1 Glucose 1.09 M

The steps of Protocol B were followed, except that glucose, 1.2755 g,was added to the stock buffer solution, 6.495 mL. This solution was pH10.5.

Protocol O2. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium CarbonatepH 9.0 Isocyanate:Amine 14.5:1 Glucose 1.09 M

The steps of Protocol B were followed, except that glucose, 1.2755 g,was added to the stock buffer solution, 6.495 mL. Using concentratedhydrochloric acid, this buffer was adjusted to pH 9.00.

Protocol P1. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium Carbonate1:1 Isocyanate:Amine 14.5:1 Urea 1.5 M

The steps of Protocol B were followed, except that urea, 0.5226 g, wasadded to the stock buffer solution, 5.807 mL, nominally 1.5 M. Thissolution was pH 11.

Protocol P2. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium CarbonatepH 9.00 Isocyanate:Amine 14.5:1 Urea 1.5 M

The steps of Protocol B were followed, except that urea, 0.5226 g, wasadded to the stock buffer solution, 5.807 mL. Using concentratedhydrochloric acid, this buffer was adjusted to pH 9.00.

Protocol Q. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 7:3

The steps of Protocol B were followed, except that the volume of theHMDI setting agent was reduced to 1.0 μL.

Protocol R. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 6.7; 6.5; 6.1:1

The steps of Protocol B were followed, except that the HMDI settingagent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with toluene. The volumeof diluted HMDI setting agent applied to the lap joint was 2 μL, 4 μL,or 6 μL, respectively.

Protocol S. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 6.1; 5.7; 5.6:1

The steps of Protocol B were followed, except that the HMDI settingagent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with methylcyclohexane.The volume of diluted HMDI setting agent applied to the lap joint was 2μL, 4 μL, or 6 μL, respectively.

Protocol T. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 8.5; 9.4; 9.7:1

The steps of Protocol B were followed, except that the HMDI settingagent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with chloroform. Thevolume of diluted HMDI setting agent applied to the lap joint was 2 μL,4 μL, or 6 μL, respectively.

Protocol U. SE0K 17% w/w Lysine hydrochloride 1:2 Potassium carbonate1:1 Isocyanate:Amine 8.0; 8.7; 7.8:1

The steps of Protocol B were followed, except that the HMDI settingagent was diluted 1:1 w/w, 1:3 w/w, or 1:5 w/w with methylene chloride.The volume of diluted HMDI setting agent applied to the lap joint was 2μL, 4 μL, or 6 μL, respectively.

Protocol V1. SE8K 33% w/w Lysine hydrochloride 1:1 Potassium carbonate3:2 Isocyanate:Amine 5.0:1

Preparation of Protein Dope

A stock solution of buffer was prepared by dissolving lysinehydrochloride, 9.14 mg/mL, potassium carbonate 41.6 mg/mL, and EvansBlue dye, 0.50 mg/mL deionized water. The mixture was filtered through aglass wool plug before use. SE8K, 113.9 mg, and 227.8 mg of stock bufferwere placed in a Eppendorf vial and agitated on a vortex mixer untildissolved. Bubbles were removed from this solution by centrifugation at5000 rpm for 30 seconds. This protein dope solution was then loaded intoa 1.00 mL syringe and let stand for 20 minutes at room temperaturebefore dispensing to the lap joint test specimens.

Preparation of HMDI Setting Agent

The HMDI setting agent was prepared by dissolving Sudan Red dye, 3.75mg, in Pluronic surfactant L-61, and adding an equal weight of1,6-diisocyanatohexane.

Preparation of Rat Skins

Freshly harvested rat hides were stored frozen at −20° C. Just beforeuse the hides were thawed and cut into 1 cm×3 cm strips. Strips wereselected which were uniform in width and thickness and which were devoidof loose fascia and muscle tissue. These rat skin samples weretemporarily stored at 37° C. between gauze pads soaked with PBS andcontained in a plastic bag to prevent drying prior to use.

Application of Adhesive Protein dope, 35 μL, was applied to one end of arat skin and worked into about a 1 cm² area with 5-10 strokes of astainless steel spatula. The excess protein dope was transferred withthe stainless steel spatula to the second strip of rat skin and workedin similarity. A total of 3.8 μL of HMDI setting agent was applied tothe skins, apportioned so that 3 parallel stripes of HMDI were appliedto the first skin. The skins were immediately assembled to form the lapjoint, rubbed against each other to distribute the HMDI setting agent,covered with a piece of plastic film to prevent drying, and compressedunder a 100 g weight. The joint was allowed to cure for 15 minutes at37° C. The length and width of the lap joint was measured to 1 mm usinga ruler immediately before tensile testing on an Instron Model 55 testmachine. The crosshead speed was set at 25 mm per minute. Lap sheartensile strengths were reported in units of g/cm². Means and standarddeviations were calculated for measurements conducted at least intriplicate.

Protocol V2. SE8K 33% w/w Lysine hydrochloride 1:2 Potassium carbonate3:2 Isocyanate:Amine 5.0:1

The method of Protocol VI was followed except that a stock solution ofbuffer was prepared by dissolving lysine hydrochloride, 4.59 mg/mL,potassium carbonate 31.2 mg/mL, and Evans Blue dye, 0.50 mg/mL indeionized water.

Protocol V3. SE8K 33% w/w Lysine hydrochloride 0:2 Potassium carbonate3:2 Isocyanate:Amine 5.0:1

The method of Protocol VI was followed except that a stock solution ofbuffer was prepared by dissolving potassium carbonate 13.83 mg/mL, andEvans Blue dye, 0.50 mg/mL in deionized water.

Protocol W1. SE0K 17% w/w Arginine 1:4 Potassium carbonate 1.2:1Isocyanate:Amine 13.1:1

The method of Protocol A was followed except that the buffer wasprepared using arginine, 2.4 mg/mL, potassium carbonate, 9.46 mg/mL, andEvans Blue dye, 0.50 mg/mL in deionized water. The volume of HMDIsetting agent applied to the lap joint was 2.0 μL. Only the alpha aminogroup of the arginine is assume to participate in the stoichiometry ofthe setting reaction.

Protocol W2. SE0K 17% w/w Cysteine 1:2 Potassium carbonate 1:1Isocyanate:Amine 13.1:1

The method of Protocol A was followed except that the buffer wasprepared using cysteine, 1.38 mg/mL, and potassium carbonate, 9.46mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The volume ofHMDI setting agent applied to the lap joint was 2.0 μL.

Protocol W3. SE0K 17% w/w Tyrosine 1:2 Potassium carbonate 1:1Isocyanate:Amine 13.1:1

The method of Protocol A was followed except that the buffer wasprepared using tyrosine, 2.07 mg/mL, and potassium carbonate, 9.46mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. The volume ofHMDI setting agent applied to the lap joint was 2.0 μL.

Protocol W4. SE0K 17% w/w 1,3-BDSA 1:2 Potassium carbonate 1:1Isocyanate:Amine 13.1:1

The method of Protocol A was followed except that the buffer wasprepared using 1,3-benzene disulfonic acid disodium salt monohydrate(1,3-BDSA), 3.79 mg/mL, and potassium carbonate, 9.46 mg/mL, and EvansBlue dye, 0.50 mg/mL in deionized water. The volume of HMDI settingagent applied to the lap joint was 2.0 μL.

Protocol X. SE0K 17% w/w Peptide RGRGRGKGKGK 1:2 (SEQ ID NO:35)Potassium carbonate 1:1 Isocyanate:Amine 14.5:1

The method of Protocol A was followed except that the buffer wasprepared using synthetic peptide RGRGRGKGKGK (SEQ ID NO:35), 4.4 mg/mL,potassium carbonate, 9.46 mg/mL, and Evans Blue dye, 0.50 mg/mL indeionized water. The volume of HMDI setting agent applied to the lapjoint was 2.0 μL.

Protocol Y. SE0K 17% w/w Cholinyl lysinate 1:4 Potassium carbonate1.12:1 Isocyanate:Amine 14.5:1

The method of Protocol B was followed except that the buffer wasprepared by dissolving cholinyl lysinate, 29.2 mg, potassium carbonate,123.9 mg, and Evans Blue dye, 6.0 mg, in deionized water, 13.09 mL. Thebuffer solution was filtered through a 0.45 micron syringe filter beforeuse. The protein dope was prepared by dissolving SE0K, 63.5 mg, in 310.0μL of buffer.

Protocol Z. SE0K 17% w/w AEGly 1:4 Potassium carbonate 1:1Isocyanate:Amine 14.5:1

The method of Protocol B was followed except that the buffer wasprepared using 2-aminoethyl glycinate, AEGly (see Example 1, diaminesynthesis). An aliquot, 28.9 mg, of 2-aminoethyl glycinate in water, 236mg/mL, and potassium carbonate, 48.6 mg, was dissolved in water, 5.127mL. Evans Blue dye, 2.20 mg was added, and the solution filtered througha 0.45 micron syringe filter.

Protocol AA. SE8K 17% w/w Lysine hydrochloride 1:1 Potassium carbonate1:1 Isocyanate:Amine 12.0:1

The method of Protocol VI was followed except that the buffer wasprepared using lysine hydrochloride, 3.73 mg/mL, potassium carbonate,11.33 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. Pluronicsurfactant L-61 was not added to the HMDI setting agent. The proteinpolymer used was SE8K. The volume of setting agent applied to the jointwas 2.0 μL.

Protocol AB. SE0K-CS1 17% w/w Lysine hydrochloride 0.94:2 Potassiumcarbonate 0.96:1 Isocyanate:Amine 16.4:1

The method of Protocol A was followed except that the buffer wasprepared using lysine hydrochloride, 1.84 mg/mL, potassium carbonate,8.37 mg/mL, and Evans Blue dye, 0.50 mg/mL in deionized water. Theprotein polymer used was SE0K-CS1. The volume of setting agent appliedto the joint was 2.0 μL.

Protocol AC. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5Isocyanate:Amine 7.3:1

The steps of Protocol F were followed, except that the volume of HMDIsetting agent applied to the lap joint was 1.0 μL.

Protocol AD. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5Isocyanate:Amine 7.3:1

The steps of Protocol F were followed, except that the HMDI settingagent was diluted 1:1 v/v, 1:3 v/v, or 1:5 v/v using cyclohexane. Thevolume of diluted HMDI setting agent applied to the lap joint was 2 μL,4 μL, or 6 μL, respectively.

Protocol AE. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5Isocyanate:Amine 7.3:1

The steps of Protocol F were followed, except that the HMDI settingagent was diluted 1:1 v/v, 1:3 v/v, or 1:5 v/v using1,1,1-trichloroethane. The volume of diluted HMDI setting agent appliedto the lap joint was 2 μL, 4 μL, or 6 μL, respectively.

Protocol AF. SE0K 17% w/w Lysine hydrochloride 1:2 Sodium Borate pH 9.5Isocyanate:Amine 7.3:1

The steps of Protocol F were followed, except that the HMDI settingagent was diluted 1:1 v/v, 1:3 v/v, 1:5 v/v, or 1:9 v/v using ethylacetate. The volume of diluted HMDI setting agent applied to the lapjoint was 2 μL, 4 μL, 6 82 L, or 10 μL, respectively.

Protocol AG. SE0K-CS1 17% w/w 1,3-PG 0.39:2 Potassium carbonate 0.496:1Potassium bicarbonate 0.496:1 Isocyanate:Amine 2.5:1

The methods of Protocol B were followed with the followingmodifications.

Preparation of Protein Dope

Protein polymer SE0K-CS1, 81.6 mg, was added to 311 μL of deionizedwater in an Eppendorf tube and agitated on a vortex mixter untildissolved. To this solution was added 11.43 μL of a solution of1,3-propanediyldiglycinate (1,3-PG) in water, 10% w/w; 14.73 μL of asolution of potassium carbonate in water, 10% w/w; and 9.43 μL of asolution of potassium bicarbonate in water, 10% w/w. The contents wereagain agitated on the vortex mixer until homogeneous. The solution wascentrifuged at about 5000 rpm for 30-60 seconds to separate air bubbles,and then the dope was exposed to reduced pressure, 26 in-Hg, for 30minutes.

Preparation of HMDI Setting Agent

Neat 1,6-diisocyanatohexane, 100 mg, and Sudan Red dye, 2.4 mg, weredissolved in 1-chloro-2,2,2-trifluoroethyl diflurormethyl ether, 2.342g. The volume of diluted HMDI setting agent applied to the lap joint was4 μL.

Protocol AH. SE0K 17% w/w Lysine 0:1 Potassium carbonate 1:1Isocyanate:Amine 5.0:1

Preparation of Protein Dope

Protein dope was prepared by dissolving protein polymer SE0K at 17% w/win 10 mMolar aqueous lactic acid, 0.90 mg/mL. This protein dope testedapproximately pH 3.5 with wide range pH test paper. A solution toinitiate curing was prepared by dissolving potassium carbonate, 1.66 g,in deionized water, 10 mL.

Preparation of HMDI Setting Agent

An HMDI setting agent was prepared by dissolving 1,6-diisocyanatohexane,5.04 g, Pluronic surfactant F-127, 0.0033 g, and Sudan Red dye, 0.0020 gin chloroform, 2.24 g.

Preparation of Rat Skins

Freshly harvested rat hides were stored frozen at −20° C. Just beforeuse the hides were thawed and cut into 1 cm×3 cm strips. All fascia wasremoved from the strips of skin with a razor blade. Strips of skin wereselected which were uniform in width and thickness. Prepared rat skinsamples were temporarily stored at 37° C. between gauze pads soaked withPBS and contained in a plastic bag to prevent drying.

Application of Adhesive

The strips of skins were arranged on a glass plate in a 37° C. warmroom. Protein dope, 15 μL, was worked into an approximately 1 cm² areaat the end of each of two rat skins using a stainless steel spatula, 30μL total. The HMDI setting agent, 1.0 μL, was worked into anapproximately 1 cm² area at the end of each of two rat skins using astainless steel spatula, 2.0 μL total. The potassium carbonate curingsolution, 2.0 μL, was added as 6 drops, 3 drops applied to each of thetwo rat skins and the skins immediately assembled to form a lap joint. A100 g weight was applied to the joint and the adhesive allowed to curefor 15 minutes at 37° C. The lap joint was tested to failure on anInstron tensile testing machine as described herein.

The first study employed the surfactant Pluronic L-31.

TABLE 18 Role of Pluronic L-31 Surfactant. HMDI + Lap Shear SEOK DopeK₂CO₃ L-Lys Pluronic g/cm² Protocol 1 SEOK 17% W/W 1:1 1:2 none 2143 ±328 A 2 SEOK 17% w/w (degassed) 1:1 1:2 none 2901 ± 685 B 3 SEOK 17% w/w(degassed) 1:1 1:2 HMDI/ 1248 ± 370 C L-31 1:1 (3.3% w/w wrt total dope)4 SEOK 17% w/w (degassed) + 1:1 1:2 none  745 ± 209 D1 L-31 (1.04% w/wwrt total dope) 5 SEOK 17% w/w (degassed) + 1:1 1:2 none 1499 ± 159 D2L-31 (0.24% w/w wrt total dope) (1.4% w/w wrt SEOK) 6 SEOK 17% w/w(degassed) + 1:1 1:2 HMDI/  677 ± 284 E L-31 (0.17% w/w wrt total L-311:1 dope) (1.0% w/w wrt SEOK) (3.3% w/w wrt total dope)

In the next study, various buffers were employed in conjunction withlysine as the polyfunctional group.

TABLE 19 Adhesive Performance Using 17% SELP0K (SE0K) in Various Buffersat pH 9.5, Lap Shear CV Buffer g/cm² % Protocol 1 Na₃BO₃/L-Lys (1:2)*2360 ± 475 20% F 2 K₃BO₃/L-LyS (1:2) 2037 ± 338 17% G 3 Li₂CO₃/L-Lys(1:2) 188 ± 38 20% H 4 Na₂CO₃/L-Lys (1:2) 1168 ± 274 23% I 5 K₂CO₃/L-Lys(1:2) 2393 ± 631 26% J 6 Cs₂CO₃/L-Lys (1:2) 233 ± 56 24% K 7 CaCO₃/L-Lys(1:2) 88 ± 2  2% L *The mole ratio of amino groups derived from lysineto amino groups derived from SELPOK.

In the next study various chemically unreactive and reactive additiveswere added to the formulation to determine the effect of the additiveson shear strength.

TABLE 20 Adhesive Performance Using 17% SELP0K in Carbonate Buffers withAdditives Lap Shear Buffer pH g/cm² CV Protocol 1 K₂CO₃ (2:2)/L-Lys(1:2) 10.5 2901 ± 685 24% B no additive 9.0 1617 ± 293 18% M 2 K₂CO₃(2:2)/L-Lys (1:2) 11 2538 ± 441 17% N1 plus Kl (O.043 Mole/L) 9.0  826 ±211 26% N2 3 K₂CO₃ (2:2)/L-Lys (1:2) 10.5 1896 ± 557 29% O1 plus Glucose(1.09 Mole/L) 9.0 1398 ± 358 26% O2 4 K₂CO₃ (2:2)/L-Lys (1:2) 11 2674 ±846 32% P1 plus Urea (1.50 Mole/L) 9.0 239 ± 38 16% P2

In the next study various organic solvents were employed, where thelinking agent was dissolved in the solvent prior to mixing with theaqueous red protein solution.

TABLE 21 Adhesive Performance Using HMDI Plus Volatile Diluents with 17%SELP0K in Lysine-Borate Buffer pH 9.5. Dilution Setting Agent Lap ShearCV Diluent bp Ratio [v/v] Volume g/cm² % Protocol 1 None n.a. 1:0 1 μL852 ± 173 20% AC 2 Cyclohexane 81° 1:1 2 μL  900 n.a. AD 1:3 4 μL  9531:5 6 μL 1053 3 1,1,1- 75° 1:1 2 μL 781 ± 13   2% AE Trichloro- 1:3 4 μL943 ± 119 13% ethane 1:5 6 μL 816 ± 51  4 Ethyl Acetate 77° 1:1 2 μL 869± 41   5% AF 1:3 4 μL 741 ± 296 40% 1:5 6 μL 685 ± 147 21% 1:9 10 μL 658 ± 131 20%

TABLE 22 Adhesive Performance Using HMDI Plus Volatile Diluents with 17%SELP0K in Lysine-Carbonate Buffer pH 10. Ratio Setting agent Lap ShearCV Diluent bp [w/w] Volume g/cm² % Protocol 1 None n.a. 1:0 1 μL 2713 ±234  6% Q 2 Toluene 110° 1:1 2 μL 2303 ± 502 22% R 1:3 4 μL 2295 ± 210 9% 1:5 6 μL 1178 ± 282 24% 3 Methyl 101° 1:1 2 μL 2508 ± 234  9% SCyclohexane 1:3 4 μL 2160 ± 111  5% 1:5 6 μL 1364 ± 395 29% 4 Chloroform 61° 1:1 2 μL 2075 ± 370 18% T 1:3 4 μL 2836 ± 620 22% 1:5 6 μL 1493 ±223 15% 5 Methylene  40° 1:1 2 μL 2389 ± 542 23% U Chloride 1:3 4 μL2636 ± 504 19% 1:5 6 μL 2511 ± 493 20%

In the next study various polyfunctional agents were employed using avariety of functionalities to crosslink the polymer, where thefunctionalities were symmetrical or unsymmetrical and the interveningchains were aliphatic or aromatic, with different functional groups asside chains. In some instances, the polyfunctional agents used forcrosslinking were hydrolytically unstable, having a hydrolyticallysusceptible bond in the linking chain.

TABLE 23 Adhesive Performance Using SELP8K, SELP0K or SELP0K-CS1 WithDifferent Polyfunctional Agents Protein and Polyfunctional Lap ShearBuffer Ratio* Agent Tensile g/cm² Protocol SE8K 33% w/w 2:2 Lysine 1328± 203 V1 K₂CO₃ 3:2 1:2 1212 ± 241 V2 0:2 116 ± 383 V3 SE0K 17% w/w 1:2Lysine 2143 ± 328 A K₂CO₃ 1:1 Arginine 1176 ± 748 W1 Cysteine 741 ± 66W2 Tyrosine 622 ± 339 W3 3,5-Disulfonato- W4 catechol 399 SE0K 17% w/w1:2 Peptide 850 ± 184 X K₂CO₃ 1:1 RGRGRGKGKGK SE0K 17% w/w 1:2 Lysine2901 ± 685 B K₂CO₃ 1:1 SE0K 17 % w/w 1:4 cholinyl lysinate 2385 ± 502 YK₂CO₃ 1.12:1 SE0K 17% w/w 1:4 2-aminoethyl glycinate 3269 ± 422 Z K₂CO₃1.12:1 N,O-Diglycyl ethanolamine 4:1 mixture as sulfate salt SE0K-CS117% 1:2 Lysine 2196 ± 275 AB w/w K₂CO₃ 1:1 SEOK-CS1 17% 0.77:21,3-Propanediyl 3028 ± 392 AG w/w Diglycinate K₂CO₃ 1:1 *Ratio of addednucleophilic groups to amino groups available on the protein backbone.

In Table 24, various formulations comprising either SELP8K, SELP0K,SELP0K-CS1 are compared.

TABLE 24 Adhesive Performance Using SELP8K, SELPOK, and SELPOK-CS1.K₂CO₃ to Amine Lap Shear Pro- Polymer Amine Ratio Ratio* Tensile g/cm² %CV tocol SE8K 1:1 2:2 2854 ± 1027 36% AA 17% w/w SE0K 1:1 1:2 2143 ±328  15% A 17% w/w SE0K 17% 1:1 0:2 532 ± 207 39% AH w/w in 10 mM aq.Lactic Acid SE0K-CS1 1:1 1:2 2196 ± 275  13% AB 17% w/w *Ratio of aminegroups derived from lysine to amine groups derived from the protein.

It is evident from the above results, that the subject inventionprovides for compositions which can set rapidly to provide compositionshaving a broad range of properties. The subject compositions can providefor strongly adhering compositions with good shear strength, the shearstrength being realized within a short period of time. The subjectinvention also provides for compositions that are capable of fillingvoids or holes in tissue or otherwise augmenting the tissue. Thus, thesubject proteinaceous polymers may be employed as tissue adhesives,providing physiologically compatible compositions which maintain theirstrength for extended periods of time, while being capable ofresorption, as well as sealants, among other uses.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

35 6 amino acids amino acid single linear peptide 1 Gly Ala Gly Ala GlySer 1 5 5 amino acids amino acid single linear peptide 2 Gly Val Gly ValPro 1 5 7 amino acids amino acid single linear peptide 3 Ala Lys Leu XaaLeu Ala Glu 1 5 64 amino acids amino acid single linear peptide 4 GlyAla Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly 20 25 30Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val 35 40 45Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 50 55 6064 amino acids amino acid single linear peptide 5 Gly Ala Gly Ala GlySer Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly AlaGly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly 20 25 30 Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val 35 40 45 Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 50 55 60 31 base pairsnucleic acid single linear cDNA 6 GCGGG TGCCTGCATG TACATCCGAG T 31 35base pairs nucleic acid single linear cDNA 7 ACTCGGATGT ACATGCAGGCACCCGCTCCA GAGCC 35 ATION FOR SEQ ID NO 8 (i) SEQUENCE CHARACTERISTICS(A) LENGTH 192 base pairs (B) TYPE nucleic acid (C) STRANDEDNESS double(D) TOPOLOGY linear (ii) MOLECULE TYPE cDNA (xi) SEQUENCE DESCRIPTIONSEQ ID NO 8 GGTGCCGGTT CTGGAGCTGG CGCGGGCTCT GGAGTAGGTG TGCCAGGTGTAGGAGTTCCG 60 GGTGTAGGCG TTCCGGGAGT TGGTGTACCT GGAGTGGGTG TTCCAGGCGTAGGTGTGCCC 120 GGGGTAGGAG TACCAGGGGT AGGCGTGCCT GGAGCGGGTG CTGGTAGCGGCGCAGGCGCG 180 GGCTCTGGAG CG 192 65 amino acids amino acid single linearpeptide 9 Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val ProGly 1 5 10 15 Val Gly Val Gly Pro Gly Val Gly Val Pro Gly Val Gly ValPro Gly 20 25 30 Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val ProGly Val 35 40 45 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala GlySer Gly 50 55 60 Ala 65 201 base pairs nucleic acid single linear cDNA10 ATGGCAGCGA AAGGGGACCG GGCTCTGGTG TTGGAGTGCC AGGTGTCGGT GTTCCGGGTG 60TAGGCGTTCC GGGAGTTGGT GTACCTGGAA AGGTGTTCCG GGGGTAGGTG TGCCGGGCGT 120TGGAGTACCA GGTGTAGGCG TCCCGGGAGC GGGTGCTGGT AGCGGCGCAG GCGCGGGCTC 180TTTCCGCTAA AGTCCTGCCG T 201 35 base pairs nucleic acid single linearcDNA 11 AAGAAGGAGA TATCATATGG CAGCGAAAGG GGACC 35 37 base pairs nucleicacid single linear cDNA 12 CGCAGATCTT TAAATTACGG CAGGACTTTA GCGGAAA 37192 base pairs nucleic acid double linear cDNA 13 GGTGCCGGTT CTGGAGCTGGCGCGGGCTCT GGTGTTGGAG TGCCAGGTGT CGGTGTTCCG 60 GGTGTAGGCG TTCCGGGAGTTGGTGTACCT GGAAAAGGTG TTCCGGGGGT AGGTGTGCCG 120 GGCGTTGGAG TACCAGGTGTAGGCGTCCCG GGAGCGGGTG CTGGTAGCGG CGCAGGCGCG 180 GGCTCTGGAG CG 192 64amino acids amino acid single linear peptide 14 Gly Ala Gly Ser Gly AlaGly Ala Gly Ser Gly Val Gly Val Pro Gly 1 5 10 15 Val Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Lys 20 25 30 Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly 35 40 45 Val Pro Gly Ala GlyAla Gly Ser Gly Ala Gly Ala Gly Ser Gly Ala 50 55 60 884 amino acidsamino acid single linear peptide 15 Met Asp Pro Val Val Leu Gln Arg ArgAsp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln Leu Asn Arg Leu Ala AlaHis Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met Gly Ala Gly Ser Gly Ala GlyAla Gly Ser Gly Val Gly Val Pro 35 40 45 Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly 50 55 60 Lys Gly Val Pro Gly Val Gly ValPro Gly Val Gly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Ala Gly AlaGly Ser Gly Ala Gly Ala Gly Ser Gly 85 90 95 Ala Gly Ala Gly Ser Gly AlaGly Ala Gly Ser Gly Val Gly Val Pro 100 105 110 Gly Val Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 Lys Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Val 130 135 140 Gly Val Pro GlyAla Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 145 150 155 160 Ala GlyAla Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 165 170 175 GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180 185 190Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 195 200205 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 210215 220 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro225 230 235 240 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly ValPro Gly 245 250 255 Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly ValPro Gly Val 260 265 270 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala GlyAla Gly Ser Gly 275 280 285 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly SerGly Val Gly Val Pro 290 295 300 Gly Val Gly Val Pro Gly Val Gly Val ProGly Val Gly Val Pro Gly 305 310 315 320 Lys Gly Val Pro Gly Val Gly ValPro Gly Val Gly Val Pro Gly Val 325 330 335 Gly Val Pro Gly Ala Gly AlaGly Ser Gly Ala Gly Ala Gly Ser Gly 340 345 350 Ala Gly Ala Gly Ser GlyAla Gly Ala Gly Ser Gly Val Gly Val Pro 355 360 365 Gly Val Gly Val ProGly Val Gly Val Pro Gly Val Gly Val Pro Gly 370 375 380 Lys Gly Val ProGly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 385 390 395 400 Gly ValPro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 405 410 415 AlaGly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 420 425 430Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 435 440445 Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 450455 460 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly465 470 475 480 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val GlyVal Pro 485 490 495 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly 500 505 510 Lys Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Val 515 520 525 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly AlaGly Ala Gly Ser Gly 530 535 540 Ala Gly Ala Gly Ser Gly Ala Gly Ala GlySer Gly Val Gly Val Pro 545 550 555 560 Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly 565 570 575 Lys Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Val 580 585 590 Gly Val Pro Gly Ala GlyAla Gly Ser Gly Ala Gly Ala Gly Ser Gly 595 600 605 Ala Gly Ala Gly SerGly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 610 615 620 Gly Val Gly ValPro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 625 630 635 640 Lys GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 645 650 655 GlyVal Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 660 665 670Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro 675 680685 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 690695 700 Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val705 710 715 720 Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala GlySer Gly 725 730 735 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly ValGly Val Pro 740 745 750 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly ValGly Val Pro Gly 755 760 765 Lys Gly Val Pro Gly Val Gly Val Pro Gly ValGly Val Pro Gly Val 770 775 780 Gly Val Pro Gly Ala Gly Ala Gly Ser GlyAla Gly Ala Gly Ser Gly 785 790 795 800 Ala Gly Ala Gly Ser Gly Ala GlyAla Gly Ser Gly Val Gly Val Pro 805 810 815 Gly Val Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly 820 825 830 Lys Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val 835 840 845 Gly Val Pro Gly AlaGly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 850 855 860 Ala Gly Ala MetAsp Pro Gly Arg Tyr Gln Asp Leu Arg Ser His His 865 870 875 880 His HisHis His 1065 amino acids amino acid single linear protein 16 Met Asp ProVal Val Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr GlnLeu Asn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met GlyAla His Gly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala 35 40 45 Gly ProLys Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly 50 55 60 Pro AlaGly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 65 70 75 80 GlnGly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 85 90 95 GlyAla Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 100 105 110Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 115 120125 Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 130135 140 Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly145 150 155 160 Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro 165 170 175 Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala 180 185 190 Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala Gln Gly 195 200 205 Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro Gly Gly Ala 210 215 220 Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala Gly Pro Gly 225 230 235 240 Gly Ala Gln Gly Pro Ala Gly ProGly Gly Ala Gln Gly Pro Ala Gly 245 250 255 Pro Gly Gly Ala Gln Gly ProAla Gly Pro Gly Gly Ala His Gly Pro 260 265 270 Ala Gly Pro Lys Gly AlaHis Gly Pro Ala Gly Pro Lys Gly Ala His 275 280 285 Gly Pro Ala Gly ProLys Gly Ala His Gly Pro Ala Gly Pro Lys Gly 290 295 300 Ala Gln Gly ProAla Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 305 310 315 320 Gly GlyAla Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 325 330 335 GlyPro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly 340 345 350Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala 355 360365 Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly 370375 380 Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly385 390 395 400 Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro 405 410 415 Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala Gln 420 425 430 Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro Gly Gly 435 440 445 Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala Gly Pro 450 455 460 Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala Gln Gly Pro Ala 465 470 475 480 Gly Pro Gly Gly Ala Gln Gly ProAla Gly Pro Gly Gly Ala Gln Gly 485 490 495 Pro Ala Gly Pro Gly Gly AlaGln Gly Pro Ala Gly Pro Gly Gly Ala 500 505 510 Gln Gly Pro Ala Gly ProGly Gly Ala His Gly Pro Ala Gly Pro Lys 515 520 525 Gly Ala His Gly ProAla Gly Pro Lys Gly Ala His Gly Pro Ala Gly 530 535 540 Pro Lys Gly AlaHis Gly Pro Ala Gly Pro Lys Gly Ala Gln Gly Pro 545 550 555 560 Ala GlyPro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 565 570 575 GlyPro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 580 585 590Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro 595 600605 Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala 610615 620 Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly625 630 635 640 Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala 645 650 655 Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro Gly 660 665 670 Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala Gly 675 680 685 Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala Gln Gly Pro 690 695 700 Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro Gly Gly Ala Gla 705 710 715 720 Gly Pro Ala Gly Pro Gly Gly AlaGln Gly Pro Ala Gly Pro Gly Gly 725 730 735 Ala Gln Gly Pro Ala Gly ProGly Gly Ala Gln Gly Pro Ala Gly Pro 740 745 750 Gly Gly Ala Gln Gly ProAla Gly Pro Gly Gly Ala Gln Gly Pro Ala 755 760 765 Gly Pro Gly Gly AlaHis Gly Pro Ala Gly Pro Lys Gly Ala His Gly 770 775 780 Pro Ala Gly ProLys Gly Ala His Gly Pro Ala Gly Pro Lys Gly Ala 785 790 795 800 His GlyPro Ala Gly Pro Lys Gly Ala Gln Gly Pro Ala Gly Pro Gly 805 810 815 GlyAla Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly 820 825 830Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro 835 840845 Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln 850855 860 Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly865 870 875 880 Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro 885 890 895 Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala 900 905 910 Gly Pro Gly Gly Ala Gln Gly Pro Ala Gly Pro GlyGly Ala Gln Gly 915 920 925 Pro Ala Gly Pro Gly Gly Ala Gln Gly Pro AlaGly Pro Gly Gly Ala 930 935 940 Gln Gly Pro Ala Gly Pro Gly Gly Ala GlnGly Pro Ala Gly Pro Gly 945 950 955 960 Gly Ala Gln Gly Pro Ala Gly ProGly Gly Ala Gln Gly Pro Ala Gly 965 970 975 Pro Gly Gly Ala Gln Gly ProAla Gly Pro Gly Gly Ala Gln Gly Pro 980 985 990 Ala Gly Pro Gly Gly AlaGln Gly Pro Ala Gly Pro Gly Gly Ala Gln 995 1000 1005 Gly Pro Ala GlyPro Gly Gly Ala Gln Gly Pro Ala Gly Pro Gly Gly 1010 1015 1020 Ala HisGly Pro Ala Gly Pro Lys Gly Ala His Gly Pro Ala Gly Pro 1025 1030 10351040 Lys Gly Ala Met Asp Pro Gly Arg Tyr Gln Leu Ser Ala Gly Arg Tyr1045 1050 1055 His Tyr Gln Leu Val Trp Cys Cys Lys 1060 1065 4 aminoacids amino acid single linear peptide 17 Pro Leu Gly Pro 1 22 aminoacids amino acid single linear peptide 18 Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro Leu Gly Pro Leu Gly 1 5 10 15 Pro Gly Val Gly Val Pro 2024 amino acids amino acid single linear peptide 19 Gly Ala Gly Ala GlySer Gly Val Gly Val Pro Gly Phe Phe Val Arg 1 5 10 15 Ala Arg Arg GlyVal Gly Val Pro 20 32 base pairs nucleic acid single linear othernucleic acid /desc = “synthetic” 20 GATCTTCGAT CTCATCACCA TCACCATCAC TA32 32 base pairs nucleic acid single linear other nucleic acid /desc =“synthetic” 21 TGCTTAGTGA TGGTGATGGT GATGAGATCG AA 32 93 base pairsnucleic acid single linear other nucleic acid /desc = “synthetic” 22ATGGCAGCGA AAGGGGACCG GTGCCGGCGC AGGTAGCGGA GCCGGTGCGG GCTCAAAAAG 60GGCTCTGGTG CCTTTCCGCT AAAGTCCTGC CGT 93 162 base pairs nucleic aciddouble linear other nucleic acid /desc = “synthetic” 23 GGGCTCTGGTGTTGGAGTGC CAGGTGTCGG TGTTCCGGGT GTAGGCGTTC CGGGAGTTGG 60 TGTACCTGGAAAAGGTGTTC CGGGGGTAGG TGTGCCGGGC GTTGGAGTAC CAGGTGTAGG 120 CGTCCCGGGAGCGGGTGCTG GTAGCGGCGC AGGCGCGGGC TC 162 54 amino acids amino acid singlelinear peptide 24 Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro GlyVal Gly Val 1 5 10 15 Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro GlyVal Gly Val Pro 20 25 30 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly AlaGly Ala Gly Ser 35 40 45 Gly Ala Gly Ala Gly Ser 50 1002 amino acidsamino acid single linear protein 25 Met Asp Pro Val Val Leu Gln Arg ArgAsp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln Leu Asn Arg Leu Ala AlaHis Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met Gly Ala Gly Ala Gly Ser GlyAla Gly Ala Gly Ser Gly Val Gly 35 40 45 Val Pro Gly Val Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val 50 55 60 Pro Gly Lys Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro 65 70 75 80 Gly Val Gly Val Pro Gly AlaGly Ala Gly Ser Gly Ala Gly Ala Gly 85 90 95 Ser Gly Val Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly Val Gly Val Pro GlyLys Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 Val Gly Val Pro GlyVal Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 130 135 140 Ala Gly Ala GlySer Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 145 150 155 160 Val GlyVal Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 165 170 175 GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 180 185 190Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val 195 200205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 210215 220 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val225 230 235 240 Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly 245 250 255 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro GlyVal Gly Val 260 265 270 Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro GlyVal Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly Ala Gly Ala Gly SerGly Ala Gly Ala Gly 290 295 300 Ser Gly Val Gly Val Pro Gly Val Gly ValPro Gly Val Gly Val Pro 305 310 315 320 Gly Val Gly Val Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly 340 345 350 Ala Gly Ala Gly Ser GlyVal Gly Val Pro Gly Val Gly Val Pro Gly 355 360 365 Val Gly Val Pro GlyVal Gly Val Pro Gly Lys Gly Val Pro Gly Val 370 375 380 Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 385 390 395 400 Ala GlySer Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val 405 410 415 GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 420 425 430Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 435 440445 Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 450455 460 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val465 470 475 480 Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro 485 490 495 Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly AlaGly Ala Gly 500 505 510 Ser Gly Val Gly Val Pro Gly Val Gly Val Pro GlyVal Gly Val Pro 515 520 525 Gly Val Gly Val Pro Gly Lys Gly Val Pro GlyVal Gly Val Pro Gly 530 535 540 Val Gly Val Pro Gly Val Gly Val Pro GlyAla Gly Ala Gly Ser Gly 545 550 555 560 Ala Gly Ala Gly Ser Gly Val GlyVal Pro Gly Val Gly Val Pro Gly 565 570 575 Val Gly Val Pro Gly Val GlyVal Pro Gly Lys Gly Val Pro Gly Val 580 585 590 Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Ala Gly 595 600 605 Ala Gly Ser Gly AlaGly Ala Gly Ser Gly Val Gly Val Pro Gly Val 610 615 620 Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 625 630 635 640 Val ProGly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 645 650 655 ProGly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 660 665 670Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 675 680685 Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 690695 700 Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly705 710 715 720 Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro 725 730 735 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val GlyVal Pro Gly 740 745 750 Val Gly Val Pro Gly Val Gly Val Pro Gly Ala GlyAla Gly Ser Gly 755 760 765 Ala Gly Ala Gly Ser Gly Val Gly Val Pro GlyVal Gly Val Pro Gly 770 775 780 Val Gly Val Pro Gly Val Gly Val Pro GlyLys Gly Val Pro Gly Val 785 790 795 800 Gly Val Pro Gly Val Gly Val ProGly Val Gly Val Pro Gly Ala Gly 805 810 815 Ala Gly Ser Gly Ala Gly AlaGly Ser Gly Val Gly Val Pro Gly Val 820 825 830 Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Lys Gly 835 840 845 Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val 850 855 860 Pro Gly Ala GlyAla Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 865 870 875 880 Val ProGly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 885 890 895 ProGly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 900 905 910Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 915 920925 Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 930935 940 Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly945 950 955 960 Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala GlySer Gly 965 970 975 Ala Gly Ala Gly Ser Gly Ala Gly Ala Met Asp Pro GlyArg Tyr Gln 980 985 990 Asp Leu Arg Ser His His His His His His 995 1000378 amino acids amino acid single linear protein 26 Met Asp Pro Val ValLeu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln Leu AsnArg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met Gly Ala GlyAla Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 35 40 45 Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 50 55 60 Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 65 70 75 80 Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 85 90 95 Ser Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 100 105 110 Gly ValGly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly 115 120 125 ValGly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 130 135 140Ala Gly Ala Gly Ser Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 145 150155 160 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val165 170 175 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly AlaGly 180 185 190 Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val ProGly Val 195 200 205 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val ProGly Lys Gly 210 215 220 Val Pro Gly Val Gly Val Pro Gly Val Gly Val ProGly Val Gly Val 225 230 235 240 Pro Gly Ala Gly Ala Gly Ser Gly Ala GlyAla Gly Ser Gly Val Gly 245 250 255 Val Pro Gly Val Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val 260 265 270 Pro Gly Lys Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro 275 280 285 Gly Val Gly Val Pro Gly AlaGly Ala Gly Ser Gly Ala Gly Ala Gly 290 295 300 Ser Gly Val Gly Val ProGly Val Gly Val Pro Gly Val Gly Val Pro 305 310 315 320 Gly Val Gly ValPro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly 325 330 335 Val Gly ValPro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly 340 345 350 Ala GlyAla Gly Ser Gly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln 355 360 365 AspLeu Arg Ser His His His His His His 370 375 141 base pairs nucleic acidsingle linear other nucleic acid /desc = “synthetic” 27 ATGGCAGCGAAAGGGGACCG CCGGTGCGGG CTCTGGTGTT GGAGTGCCGC TGGGTCCTCT 60 TGGCCCAGGTGTCGGTGTTC CGGGTGTAGG CGTTCCGGGA GTTGGTGTAC CTGGAAAAGG 120 TTTCCGCTAAAGTCCTGCCG T 141 181 base pairs nucleic acid single linear other nucleicacid /desc = “synthetic” 28 GGGCTCTGGT GTTGGAGTGC CGCTGGGTCC TCTTGGCCCAGGTGTCGGTG TTCCGGGTGT 60 AGGCGTTCCG GGAGTTGGTG TACCTGGAAA AGGTGTTCCGGGGGTAGGTG TGCCGGGCGT 120 TGGAGTACCA GGTGTAGGCG TCCCGGGAGC GGGTGCTGGTAGCGGCGCAG GCGCGGGCTC 180 T 181 60 amino acids amino acid single linearpeptide 29 Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly ValGly 1 5 10 15 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly LysGly Val 20 25 30 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro 35 40 45 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser 50 5560 936 amino acids amino acid single linear protein 30 Met Asp Pro ValVal Leu Gln Arg Arg Asp Trp Glu Asn Pro Gly Val 1 5 10 15 Thr Gln LeuAsn Arg Leu Ala Ala His Pro Pro Phe Ala Ser Asp Pro 20 25 30 Met Gly AlaGly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly 35 40 45 Val Pro LeuGly Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly 50 55 60 Val Pro GlyVal Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val 65 70 75 80 Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly 85 90 95 Ser GlyAla Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu 100 105 110 GlyPro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 115 120 125Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 130 135140 Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 145150 155 160 Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly Val GlyVal 165 170 175 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys GlyVal Pro 180 185 190 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly 195 200 205 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro 210 215 220 Leu Gly Pro Leu Gly Pro Gly Val Gly Val ProGly Val Gly Val Pro 225 230 235 240 Gly Val Gly Val Pro Gly Lys Gly ValPro Gly Val Gly Val Pro Gly 245 250 255 Val Gly Val Pro Gly Val Gly ValPro Gly Ala Gly Ala Gly Ser Gly 260 265 270 Ala Gly Ala Gly Ser Gly ValGly Val Pro Leu Gly Pro Leu Gly Pro 275 280 285 Gly Val Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly 290 295 300 Lys Gly Val Pro GlyVal Gly Val Pro Gly Val Gly Val Pro Gly Val 305 310 315 320 Gly Val ProGly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 325 330 335 Val GlyVal Pro Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly 340 345 350 ValGly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 355 360 365Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 370 375380 Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly 385390 395 400 Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro GlyVal 405 410 415 Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro GlyVal Gly 420 425 430 Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly SerGly Ala Gly 435 440 445 Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro LeuGly Pro Gly Val 450 455 460 Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Lys Gly 465 470 475 480 Val Pro Gly Val Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val 485 490 495 Pro Gly Ala Gly Ala Gly Ser GlyAla Gly Ala Gly Ser Gly Val Gly 500 505 510 Val Pro Leu Gly Pro Leu GlyPro Gly Val Gly Val Pro Gly Val Gly 515 520 525 Val Pro Gly Val Gly ValPro Gly Lys Gly Val Pro Gly Val Gly Val 530 535 540 Pro Gly Val Gly ValPro Gly Val Gly Val Pro Gly Ala Gly Ala Gly 545 550 555 560 Ser Gly AlaGly Ala Gly Ser Gly Val Gly Val Pro Leu Gly Pro Leu 565 570 575 Gly ProGly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 580 585 590 ProGly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 595 600 605Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 610 615620 Ser Gly Val Gly Val Pro Leu Gly Pro Leu Gly Pro Gly Val Gly Val 625630 635 640 Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly ValPro 645 650 655 Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly ValPro Gly 660 665 670 Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly ValGly Val Pro 675 680 685 Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro GlyVal Gly Val Pro 690 695 700 Gly Val Gly Val Pro Gly Lys Gly Val Pro GlyVal Gly Val Pro Gly 705 710 715 720 Val Gly Val Pro Gly Val Gly Val ProGly Ala Gly Ala Gly Ser Gly 725 730 735 Ala Gly Ala Gly Ser Gly Val GlyVal Pro Leu Gly Pro Leu Gly Pro 740 745 750 Gly Val Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly 755 760 765 Lys Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val 770 775 780 Gly Val Pro Gly AlaGly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly 785 790 795 800 Val Gly ValPro Leu Gly Pro Leu Gly Pro Gly Val Gly Val Pro Gly 805 810 815 Val GlyVal Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val 820 825 830 GlyVal Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly 835 840 845Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly Val Gly Val Pro Leu Gly 850 855860 Pro Leu Gly Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val 865870 875 880 Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly ValGly 885 890 895 Val Pro Gly Val Gly Val Pro Gly Ala Gly Ala Gly Ser GlyAla Gly 900 905 910 Ala Gly Ser Gly Ala Gly Ala Met Asp Pro Gly Arg TyrGln Asp Leu 915 920 925 Arg Ser His His His His His His 930 935 147 basepairs nucleic acid single linear other nucleic acid /desc = “synthetic”31 ATGGCAGCGA AAGGGGACCG CCGGTGCGGG CTCTGGTGTT GGAGTGCCAG GCTTCTTTGT 60ACGTGCACGC CGTGGTGTCG GTGTTCCGGG TGTAGGCGTT CCGGGAGTTG GTGTACCTGG 120AAAAGGTTTC CGCTAAAGTC CTGCCGT 147 186 base pairs nucleic acid singlelinear other nucleic acid /desc = “synthetic” 32 GGGCTCTGGT GTTGGAGTGCCAGGCTTCTT TGTACGTGCA CGCCGTGGTG TCGGTGTTCC 60 GGGTGTAGGC GTTCCGGGAGTTGGTGTACC TGGAAAAGGT GTTCCGGGGG TAGGTGTGCC 120 GGGCGTTGGA GTACCAGGTGTAGGCGTCCC GGGAGCGGGT GCTGGTAGCG GCGCAGGCGC 180 GGGCTC 186 62 aminoacids amino acid single linear peptide 33 Gly Ser Gly Val Gly Val ProGly Phe Phe Val Arg Ala Arg Arg Gly 1 5 10 15 Val Gly Val Pro Gly ValGly Val Pro Gly Val Gly Val Pro Gly Lys 20 25 30 Gly Val Pro Gly Val GlyVal Pro Gly Val Gly Val Pro Gly Val Gly 35 40 45 Val Pro Gly Ala Gly AlaGly Ser Gly Ala Gly Ala Gly Ser 50 55 60 966 amino acids amino acidsingle linear protein 34 Met Asp Pro Val Val Leu Gln Arg Arg Asp Trp GluAsn Pro Gly Val 1 5 10 15 Thr Gln Leu Asn Arg Leu Ala Ala His Pro ProPhe Ala Ser Asp Pro 20 25 30 Met Gly Ala Gly Ala Gly Ser Gly Ala Gly AlaGly Ser Gly Val Gly 35 40 45 Val Pro Gly Phe Phe Val Arg Ala Arg Arg GlyVal Gly Val Pro Gly 50 55 60 Val Gly Val Pro Gly Val Gly Val Pro Gly LysGly Val Pro Gly Val 65 70 75 80 Gly Val Pro Gly Val Gly Val Pro Gly ValGly Val Pro Gly Ala Gly 85 90 95 Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro Gly Phe 100 105 110 Phe Val Arg Ala Arg Arg Gly Val GlyVal Pro Gly Val Gly Val Pro 115 120 125 Gly Val Gly Val Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly 130 135 140 Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly 145 150 155 160 Ala Gly Ala Gly SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala 165 170 175 Arg Arg Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 180 185 190 Pro Gly LysGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 195 200 205 Gly ValGly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 210 215 220 SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val 225 230 235240 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 245250 255 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val260 265 270 Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly ValGly 275 280 285 Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly ValPro Gly 290 295 300 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly ValPro Gly Val 305 310 315 320 Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly 325 330 335 Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro Gly Phe 340 345 350 Phe Val Arg Ala Arg Arg Gly Val GlyVal Pro Gly Val Gly Val Pro 355 360 365 Gly Val Gly Val Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly 370 375 380 Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly 385 390 395 400 Ala Gly Ala Gly SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala 405 410 415 Arg Arg Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 420 425 430 Pro Gly LysGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 435 440 445 Gly ValGly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 450 455 460 SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val 465 470 475480 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 485490 495 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val500 505 510 Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly ValGly 515 520 525 Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly ValPro Gly 530 535 540 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly ValPro Gly Val 545 550 555 560 Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly 565 570 575 Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro Gly Phe 580 585 590 Phe Val Arg Ala Arg Arg Gly Val GlyVal Pro Gly Val Gly Val Pro 595 600 605 Gly Val Gly Val Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly 610 615 620 Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly 625 630 635 640 Ala Gly Ala Gly SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala 645 650 655 Arg Arg Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 660 665 670 Pro Gly LysGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 675 680 685 Gly ValGly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 690 695 700 SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val 705 710 715720 Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly 725730 735 Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val740 745 750 Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Ser Gly ValGly 755 760 765 Val Pro Gly Phe Phe Val Arg Ala Arg Arg Gly Val Gly ValPro Gly 770 775 780 Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly ValPro Gly Val 785 790 795 800 Gly Val Pro Gly Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly 805 810 815 Ala Gly Ser Gly Ala Gly Ala Gly Ser GlyVal Gly Val Pro Gly Phe 820 825 830 Phe Val Arg Ala Arg Arg Gly Val GlyVal Pro Gly Val Gly Val Pro 835 840 845 Gly Val Gly Val Pro Gly Lys GlyVal Pro Gly Val Gly Val Pro Gly 850 855 860 Val Gly Val Pro Gly Val GlyVal Pro Gly Ala Gly Ala Gly Ser Gly 865 870 875 880 Ala Gly Ala Gly SerGly Val Gly Val Pro Gly Phe Phe Val Arg Ala 885 890 895 Arg Arg Gly ValGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val 900 905 910 Pro Gly LysGly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro 915 920 925 Gly ValGly Val Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly 930 935 940 SerGly Ala Gly Ala Met Asp Pro Gly Arg Tyr Gln Asp Leu Arg Ser 945 950 955960 His His His His His His 965 11 amino acids amino acid single linearpeptide 35 Arg Gly Arg Gly Arg Gly Lys Gly Lys Gly Lys 1 5 10

What is claimed is:
 1. A crosslinked protein composition, said proteinprior to crosslinking comprising at least 70 weight % of repetitiveunits of GAGAGS (SEQ ID NO:01) and GVGVP (SEQ ID NO:02), where in atleast two units an amino acid is substituted with at least one of lysineor arginine, said protein having a lysine and arginine equivalent weightin the range of 1 to 20 kD, and said protein comprising at least twoamino acids which comprise a functional group capable of reacting with acrosslinking agent.
 2. A method of sealing a defect in viable tissue,said method comprising: injecting into said defect a precursorcomposition comprising a polyfunctional crosslinking agent and a proteinpolymer comprising at least 40 weight % of repetitive units of from 3 to15 amino acids of at least one naturally occurring structural protein,where said protein polymer comprises at least two amino acids whichcomprise a functional group capable of reacting with said crosslinkingagent, whereby said precursor composition sets up to a strongly adherentadhesive composition; whereby said defect is sealed.
 3. A methodaccording to claim 2, wherein said units are selected from the groupconsisting of GAGAGS (SEQ ID NO:01), GVGVP (SEQ ID NO:02) and GXX,wherein the X's are the same or different and X is any amino acid, atleast 10 number % and not more than 60 number % of X's being proline. 4.A method according to claim 2, wherein said units are GAGAGS (SEQ IDNO:01) and GVGVP (SEQ ID NO:02) and are present to form a blockcopolymer protein.
 5. A method according to claim 2, wherein said tissueis the wall of a vessel selected from an artery, vein, capillary, lung,dura or colon.
 6. A method according to claim 2 wherein said injectionis done to augment tissue mass.
 7. A method of filling a defect inviable tissue, said method comprising: injecting into said defect aprecursor composition comprising a polyfunctional crosslinking agent anda protein polymer comprising at least 40 weight % of repetitive units offrom 3 to 15 amino acids of at least one naturally occurring structuralprotein, where said protein polymer comprises at least two amino acidswhich comprise a functional group capable of reacting with saidcrosslinking agent, whereby said precursor composition sets up to astrongly adherent adhesive composition; and whereby said defect isfilled.
 8. A method according to claim 7 wherein said injection is doneto augment tissue mass.